Fluorescent and Chemiluminescent Techniques for Life Science Applications

FOR LIFE SCIENCE RESEARCH Volume 2 Number 5

• DNA & RNA Detection Techniques

• Fluorescent Microarray Experiments

• Fluorescence Multiplexing

• Ni-NTA-Atto™ Conjugates

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sigma.com/yfgbiofiles 1 Introduction

Sensitive and selective detection methods are fundamental to elucidate the function of biological processes within cellular networks. Luminescent techniques with fluorescent probes meet the demands required for this research, allowing scientists to track molecu- FOR LIFE SCIENCE RESEARCH lar interactions, mobility, and conformational changes of biomolecules in living cells with high temporal and spatial resolution. 2007 This issue of BioFiles focuses on Sigma’s products for fluorescent and chemiluminescent Volume 2 bioanalytical techniques including: Number 5 ■ Single molecule investigations ■ Kinetic enzyme measurements Table of Contents ■ Flow cytometry ■ Western blotting ■ Microarray screening Fluorescent and Chemiluminescent Techniques for Life Science ■ qPCR and qRT-PCR Applications Based on our extensive experience and expertise in organic synthesis and life sciences, as well as stringent quality assurance, Sigma offers a comprehensive selection of reagents for superior application results. The R&D scientists in our Detection Center of Excellence in Buchs, Switzerland, are continuously investigating and developing new QuantiGene® 2.0 RNA Quantification products and optimizing protocols for applications in the fields of detection and biochemistry in order to ensure the highest level of performance. System ...... 2 Comprehensive Quantitative ■ The Atto™ dyes are a series of fluorophores that can be used in the most sensitive PCR and qRT-PCR Products ...... 4 applications and are especially suitable for target-specific detection. In this issue, scientists from the Swiss Federal Institute of Technology ETH, Zurich, Switzerland, show extraordinary application results using Atto dyes in microarray experiments. Analyzing Properties of Fluorescent Atto dye conjugates and kits for protein labeling, polyhistidine detection, and Dyes used for Labeling DNA in Western blot multiplexing are also reviewed. Microarray Experiments ...... 5 ■ For genomic applications, the QuantiGene® 2.0 RNA Quantification System provides a unique approach for RNA detection and quantification by using branched DNA Protein Labeling Kits ...... 9 technology. The QuantiGene system expands our line of innovative products for real- time qPCR and qRT-PCR applications. Fluorescence Multiplexing using ■ Ampliflu™ Red is a new peroxidase substrate that provides easy and reliable fluores- Antibody-Atto™ Dye Conjugates ...... 10 cent detection of peroxidase-labeled antibodies. Sigma is a leading supplier of fluorogenic enzyme substrates that are convenient and consistent. Ni-NTA-Atto™ Conjugates for Sensitive, ■ The non-cytotoxic PHK linker dyes deliver stable and intense fluorescent labeling of Specific Detection of Polyhistidine- live cells for a variety of downstream applications such as in vivo cell tracking and Tagged Proteins ...... 12 cell-to-cell interactions. ■ In cooperation with the German Federal Institute for Material Research and Testing Fluorogenic Substrates for the Sensitive (BAM), Sigma has developed the Spectral Fluorescence Standard Kit. This kit allows Detection of Enzymes ...... 14 researchers to determine the relative spectral responsiveness of fluorescence instruments and compare fluorescent measurements. Ampliflu™ Red, a Fluorescent Peroxidase Substrate for Immunoblot We hope that you will find our articles and products helpful and interesting. Applications...... 18 For more information on detection products available from Sigma, please visit Certified Fluorescence Standards ...... 19 sigma.com/fluorescence. PKH Linker Kits for Fluorescent Contributing authors: Dr. Jens Sobek, Catharine Aquino, Dr. Ralph Schlapbach, Cell Labeling...... 22 Functional Genomics Center Zurich, Zurich, Switzerland, Monika Bäumle, Ph.D., Roland Meier, Alexander Rück, Ph.D., Bernhard Schönenberger, Ph.D., Roland Wohlgemuth, Fluorescent Microparticles and Ph.D., Sigma-Aldrich, Buchs, Switzerland, Leigh Gaskill, Sigma-Aldrich, St. Louis, MO, Nanobeads ...... 25 USA Books ...... 28

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Accurate and Precise Gene Expression Assay Reproducible Gene Expression Samples Validate RNAi Assays Human A549 cells were seeded in T-Flasks and grown to 80% confluency. At this time, cells were trypsinized 120 ® and reseeded into 96-well plates 24 hours prior to 2.0 RNA Quantification System infection. MISSION TRC shRNA lentiviral particles 100 were added to the appropriate wells. Three infections Infection 1 were performed, at a multiplicity of infection (MOI) of 80 Infection 2 Infection 3 1, in triplicate with eight different lentiviral particles 60 containing shRNA constructs in order to observe the variability of infection. The remaining wells were 40 treated with empty control virus (SHC001V). Viral % Expression constructs were allowed 24 hours incubation with the 20 cell lines prior to replacing with selective media. Cells 0 were harvested after growing to ~80% confluency. Gene Expression was then measured in 48 independent assays to verify precision and accuracy. Although expression varied among shRNA constructs, (SHC001V) gene expression was consistent among infections. ACP1 shRNAACP1 clone shRNA A PSPH clone shRNA B PSPH clone shRNA A Empty clone Control B Virus This experiment demonstrates the reproducibility of CDKN3 shRNACDKN3 clone shRNA A ENPP1 clone shRNA B ENPP1 clone shRNA A clone B target gene knockdown using shRNA constructs while highlighting the precision of the QuantiGene assay.

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For more information, visit our Web site at sigma.com/pcr. 5 Analyzing Properties of Fluorescent Dyes Used for Labeling DNA in Microarray Experiments

Dr. Jens Sobek, Catharine Aquino, Dr. Ralph Schlapbach, Array Production Functional Genomics Center Zurich, Zurich, Switzerland A sciFLEXARRAYER™ (Scienion, Berlin, Germany) and a Cy™3 and Cy5 are two fluorescent dyes frequently used for Piezorray™ (Perkin Elmer, Downers Grove, IL, USA) non-contact microarray analysis. These two dyes have favorable properties, dispensing instrument were used for microarray production. A including relatively high fluorescence quantum yields and a mini- dilution series (20, 10, 5, and 2.5 µM) of the three 71-mer oligo- mal spectral overlap, making them suitable for multiplex detec- nucleotides (71prox, 71cent, and 71dist) in 33 saline-sodium citrate buffer (SSC) were spotted in 15 replicates (1.5 nL per tion applications. However, the Cy dyes and derivatives have low spot) on epoxysilane coated slides (Scienion, Berlin, Germany). photostability compared to dyes that have been developed more Oligonucleotides were immobilized to the slide surface by over- recently. night incubation in a humidity chamber at room temperature.2 The object of our study is to replace Cy5 dye in the detection of arrayed oligonucleotides with dyes that (1) are more stable and Hybridization

(2) give a higher fluorescence signal in microarray experiments. Washing, blocking, hybridization, and drying steps were per- Experiments Fluorescent Microarray For this study we have developed a model system that allows us formed in an automated hybridization station (HS4800, Tecan, to investigate the fluorescence and stability properties of oligo- Salzburg, Austria), allowing 12 slides to be hybridized in parallel. nucleotide-dye conjugates hybridized to the slide surface without 50 nM solutions of the 13-mer oligonucleotide-dye conjugates laborious, time-consuming sample preparation. We reduced the were hybridized to four replicate slides at 30 °C in HEPES/SSC/SDS complexity of the experiment by spotting only a few oligonucle- for 2 hours. After hybridization, the slides were washed under otides and by hybridizing a single dye-labeled complementary non-stringent conditions at 23 °C sequentially with 23 SSC / oligonucleotide to each slide. Here we present preliminary results 0.2% SDS, 0.23 SSC / 0.2% SDS, and 0.23 SSC. The slides were that compare the established blue indocarbocyanine dyes Cy5, DY- then dried with nitrogen in the hybridization station to avoid 647, and Alexa Fluor® 647, to promising candidates Atto 633 and air contact. After drying, the slides were quickly transferred to a Atto 647N. The results of the complete study will be published plastic bag filled with nitrogen to reduce the time of air exposure elsewhere.1 to 10-15 seconds. Once in the bag, the slides were covered with blank glass slides to protect the dyes at the microarray surface from degradation by ozone and other oxidizing agents present in Experimental Details the air.

Determination of Dye Stability Materials To determine dye stability, the slides were scanned 12 times under Three 71-mer oligonucleotides (Operon, Cologne, Germany) that a nitrogen atmosphere. Data provided by a nearby environmental share a common 13-mer binding region were used in this study. measuring station (Stampfenbachplatz, Zurich) were used to esti- The common 13-mer region is located at the proximal 3’ end mate the atmospheric ozone concentration. For a correlation with (71prox), near the center (71cent), or at the distal 5’ end (71dist) these values, the concentration of atmospheric ozone in our labo- of the oligonucleotide. 13-mer oligonucleotide-dye conjugates ratory was measured once using a spectrophotometer.3 We found (5’-dye-TGCCTGAAGCTAT; ON-Dye) that contain the sequence that the ozone concentration in our laboratory was ~75% of the (ON) complementary to the binding region in the 71-mers were concentration measured at Stampfenbachplatz.4 synthesized by Proligo (Hamburg, Germany) (Atto 633, Atto 647N), Microsynth (Balgach, Switzerland) (Cy5), Dyomics (Jena, Scanning and Data Analysis Germany) (DY-647), and IBA (Göttingen, Germany) (Alexa Fluor All slides were scanned under identical conditions using a Tecan 647). All dyes except Cy5 were incorporated as the NHS ester via LS400 laser scanner. Arrays were protected by a cover glass and an aminohexyl linker in a separate reaction following oligonucle- scanned with the hybridized side facing downwards. The scan- otide synthesis. For incorporation of Cy5, the phosphoramidite ner focus was set to the lower side of the slide and fluorescence building block was used as received from Glen Research (Sterling, was measured through the glass. Scans were evaluated using VA, USA). Water was deionized and filtered using a Milli-Q® ArrayPro™ Analyzer 4.5.1 software (Media Cybernetics, Silver Synthesis A10 purification system (Millipore, Billerica, MA, USA). Spring, MD, USA). Reported mean fluorescence intensity values All oligonucleotides were purified by HPLC. Sodium chloride are calculated from four replicate slides. (molecular biology grade), HEPES (N-[2-hydroxyethyl]piperazine- N`-[2-ethanesulfonic acid], >99.5%), trisodium citrate (>99%), hydrochloric acid (p.a.), and sodium dodecyl sulfate (>98%) were Absorption and Fluorescence Spectra obtained from Sigma (Buchs, Switzerland). Absorption and fluorescence spectra were taken in 13 SSC using a Tecan Safire2™ spectrometer. Fluorescence was excited at 600 nm and detected at the surface of the solution (top reading

mode). sigma-aldrich.com/biofiles 6 Analyzing Properties of Fluorescent Dyes Used for Labeling DNA in Microarray Experiments

Fluorescence Intensities A typical image of a slide hybridized with Atto 647N is shown in Figure 3. The fluorescence signals for each of the oligonucleotide-dye conjugates after hybridization to the three 71-mer oligonucleotide probes were measured on four replicate slides and averaged (see Figure 4). The analysis of the fluorescence Indocarbocyanines Carborhodamines data shows a non-uniform pattern. The most intense signals were observed for the oligonucleotide-dye conjugates ON-Cy5 and ON- Figure 1. Chemical structures of indocarbocyanines and carborhodamines dyes. Atto 647N. The oligonucleotide-dye conjugates ON-DY-647, ON- Alexa Fluor 647, and ON-Atto 633, show significantly lower signals when hybridized to the 71dist and 71cent oligonucleotides; R1 R2 R3 R4 these fluorescence intensities are sequence-dependent. Nazarenko, Cy5 - (CH ) -OH (CH ) -COOH - 2 3 2 3 et al., observed similar sequence-dependant fluorescence proper- - DY-647 (CH2)3-COOH C2H5 C2H5 SO3 ties of labeled oligonucleotides when hybridized to a complemen- 6 Alexa - - - tary strand in solution. The authors noted that the effect was (CH2)3-COOH (CH2)3-SO3 (CH2)3-SO3 SO3 Fluor 647 greatest when a guanine base was present in direct proximity to the dye in the complementary strand. Since guanine has the small- Results and Discussion est oxidation potential of the four DNA nucleotides,7 the authors Molecular structures of the dyes evaluated are shown in Figure 1.

Experiments attributed the effect to fluorescence quenching by electron trans- The core chromophore structure is identical for the classical indo- fer. The relative fluorescence intensities measured in our experi- carbocyanine dyes Cy5, DY-647, and Alexa Fluor 647. The com- ment correlate with the overall dye charge. Dyes with a higher pounds differ by the types and location of alkyl groups and the negative charge demonstrated stronger fluorescence quenching. A number of sulfonic acid groups present, which affects the overall 1 charge of the core (+1, -1, and -3, respectively, for the three dyes). detailed analysis of the observed effects will be given elsewhere.

Fluorescent Microarray Microarray Fluorescent Atto 633 and Atto 647N belong to the class of carborhodamine 20 µM 10 µM 5 µM 2.5 µM dyes, and both carry a charge of +1.5 Absorbance and fluorescence spectra for all five dyes are shown in Figure 2. An analysis of the spectra will be given in a forthcoming publication.1 71dist A 260000

ON-Cy5 ON-DY-647 195000 ON-Alexa Fluor 647 ON-Atto 633 71cent ON-Atto 647N 130000

65000

71prox

Molar Absorbance Coefficient/L/mol cm Molar Absorbance Coefficient/L/mol 0 300 400 500 600 700 Wavelength/nm Figure 3. Fluorescence image measured with a confocal microscope. The spot-to-spot distance is 500 µm.

B 50000 Cy5 DY-647 40000 Alexa Fluor 647 Atto 633 Atto 647N 30000 in 1xSSC

lex = 600 nm 20000

10000 Relative Fluorescence Intensity

0 650 700 750 800 850

Wavelength/nm Figure 2: Absorption (A) and fluorescence (B) spectra of oligonucleotide- dye conjugates (ON-Dye). sigma-aldrich.com/biofiles 7

A Investigation of Dye Stability 40000 71prox 20 µM A limitation of many labeling dyes that absorb at long wavelengths, especially cyanine dyes including Cy5, DY-647, and Alexa Fluor 647, is an intrinsic low photostability and enhanced 30000 degradation by atmospheric ozone.8 Rapid dye degradation due to high ozone concentration during slide scanning may result in 20000 invalid data. In Zurich, ozone concentrations of 10-30 ppb and 10-75+ ppb were measured during winter and summer, respec- 4 10000 tively. A few minutes of exposure of unprotected slides at the highest ozone concentrations will result in a notable degradation of indocarbocyanine dyes. Relative Fluorescence Intensity 0 To compare the dye stability, we performed two sets of experi- 5 7 3 N y 4 3 7 Experiments Fluorescent Microarray 6 C - 6 4 ments.First, to determine photostability hybridized slides were 6 Y D tto o t scanned 12 times. Since photodegradation strongly increases A t A with increasing ozone concentration we have scanned the slides

Alexa Fluor 647 under nitrogen using a protective cover glass (see Experimental Details). Second, an alternative batch of hybridized slides was B stored under ambient laboratory conditions in the dark, exposed 60000 71cent 20 µM to air containing ozone concentrations of ~15 ppb for 30 minutes and 30 ppb for 150 minutes, respectively. Slides were scanned before and after this treatment under nitrogen.9 45000 Scanning the slides 12 times results in signal degradation between 3% and 9%, with Atto 647N demonstrating the least degradation 30000 (see Figure 5). The most prominent signal intensity decrease was observed for Cy5 and Atto 633.

15000 1.00 Relative Fluorescence Intensity 0

5 7 3 N y 4 3 7 0.95 C -6 6 4 Y o 6 t D t o t A t A

lexa Fluor 647 A 0.90

C 60000 71dist 20 µM 0.85

Ratio Relative Fluorescence Intensity Ratio Relative Fluorescence 0.0 45000 7 7 3 4 4 3 6 6 Cy5 6 - r Y o o t D u t l F A

30000 Atto 647N a x e l A 15000 Figure 5. Dye degradation by irradiation. Hybridized slides were scanned 12 times. The ratio of fluorescence intensities of the first and last scan Relative Fluorescence Intensity are plotted. To exclude the influence of ozone, slices were scanned under 0 nitrogen.

7 N 4 7 6 Cy5 - 4 6 Y D o t Atto 633 t A

Alexa Fluor 647 sigma-aldrich.com/biofiles Figure 4. Fluorescence intensities of oligonucleotide-dye conjugates (ON-Dye) after hybridization to complementary 71-mer oligonucleotide (A, 71prox, B, 71cent, or C, 71dist) immobilized on an epoxysilane coated surface. 8 Analyzing Properties of Fluorescent Dyes Used for Labeling DNA in Microarray Experiments

Exposure of cyanine dyes to ozone had a tremendous effect on Conclusions fluorescence intensity of dyes at surfaces (see Figure 6.). A We measured the relative fluorescence intensities and the 30 minute exposure of ON-Cy5 to a relatively low ozone concen- stability of 5 oligonucleotide-dye conjugates hybridized to three tration (15 ppb) results in a 30% loss in fluorescence intensity. 71-mer oligonucleotides printed on a microarray glass surface. Under these conditions, the sulfonated dyes (ON-DY-647 and We observed fluorescence intensities that strongly depend on ON-Alexa Fluor 647) are more stable. At a higher air ozone con- probe sequence for the dyes DY-647, Alexa Fluor 647, and Atto centration (30 ppb) and with prolonged exposure (150 minutes), 633. Cy5 and Atto 647N produced the strongest fluorescence the signal intensity was reduced by 75% (Alexa Fluor 647), 79% signals and were minimally influenced by the position of the probe (DY-647), and 89% (Cy5). The Atto dyes demonstrated only a sequence within the oligonucleotide. minor loss of fluorescence intensity, with a reduction of 9% for All dyes tested are sufficiently stable to repeated scanning at the Atto 647N, and of less than 1% for Atto 633. We even observed excitation wavelength when protected from atmospheric ozone. an initial increase in fluorescence intensity for the carborhoda- Atto 633 and Atto 647N showed minor loss of fluorescence signal mine-based Atto dyes. An explanation for this observation will be on exposure to ozone, making their use as labeling dyes in micro- given elsewhere.1 array applications optimal from this point of view. A We are aware that the results of this study do not indicate how successfully these dyes may be used in secondary DNA labeling 1.4 reactions with aminoallyl substituted nucleotides and subsequent hybridization. We are currently performing experiments that com- 1.2 pare the properties of indocarbocyanine dyes to carborhodamine Experiments dyes in labeling DNA samples and hybridization in biological sys- 1.0 tems.

0.8 References and Comments: 1. Sobek, J., Aquino, C., Schlapbach, R., to be published.

Fluorescent Microarray Microarray Fluorescent 0.6 2. Sobek, J., Aquino, C., Schlapbach, R., Quality Considerations and Selection of Surface Chemistry for Glass-Based DNA, Peptide, Antibody, Carbohydrate, and Small Molecule Microarrays. Microarrays. Volume II,

Ratio Relative Fluorescence Intensity 0.0 Applications and Data Analysis Series: Methods in Molecular Biology. 5 7 3 y 7 N 4 3 7 Rampal, J.B. (Ed.), 2nd ed., Humana Press (2007). C 6 4 6 - 6 4 Y o 6 r t 3. Many thanks to Dr. Jürg Brunner (Environment and Health Protection of D o t o t lu A t F the City of Zurich) for performing the measurements. A a x 4. www.stadtzuerich.ch/internet/ugz/home/fachbereiche/luftqualitaet/ le A messwerte/tagesverlauf.html 5. Thanks to Prof. Karl-Heinz Drexhage, ATTO-Tec (Siegen, Germany), for information on the compounds. B 6. Nazarenko, I., Pires, R., Lowe, B., Obaidy, M., Rashtchian, A., Effect of primary and secondary structure of oligodeoxyribonucleotides on the 1.0 fluorescent properties of conjugated dyes. Nucleic Acids Res., 30(9), 2089-2195 (2002). 0.8 7. Seidel, C.A.M., et al., Nucleobase-specific quenching of fluorescent dyes. 1. Nucleobase one-electron redox potentials and their correlation with static and dynamic quenching efficiencies. J. Phys. Chem., 100, 5541-5553 0.6 (1996). 8. Fare, T.L., et al., Effects of atmospheric ozone on microarray data quality. 0.4 Anal. Chem., 75, 4672-4675 (2003). 9. The dyes are stable for several weeks when the slides are stored in the 0.2 dark under a nitrogen atmosphere.

0.0 Ratio Relative Fluorescence Intensity Ratio Relative Fluorescence

5 3 y 7 3 C 4 6 6 o r t DY-647 o t lu A tto 647N F A a x le A Figure 6. Influence of atmospheric ozone on fluorescence intensity. Hybridized slides were exposed in the dark to ozone concentrations of A) 15 ppb (30 µg/m3) for 30 minutes, and B) 25 ppb (50 µg/m3) for 150 minutes. Intensities were measured before and after exposure. sigma-aldrich.com/biofiles 9 Protein Labeling

Protein Labeling Kits Dye Molar Ratio Dye/Antibody RFI* Sigma’s new protein labeling kits provide an easy and reliable Atto 550 2.0 6,000 way to label purified proteins, enzymes, and antibodies. The kits Alexa Fluor 555 3.0 1,500 include a high quality Atto dye and contain all components nec- essary to rapidly label and purify five protein samples. Atto dyes Cy3 3.5 3,000 have a bright fluorescent signal with a narrow fluorescence emis- Table 1. * RFI values are in terms of arbitrary units. sion spectrum. Atto dyes efficiently label proteins, and the resulting conjugates demonstrate high relative fluorescence intensity when compared to other commercially available fluorescence dyes. In a second experiment, monoclonal anti-mouse IgG was labeled Five protein labeling kits are available, each containing a different using Atto 647N, Alexa Fluor 647, or Cy5 (see Table 2). The RFI reactive Atto dye specific for a commonly used excitation wave- signals of the dye-labeled antibody solutions were detected using length: a spectrofluorimeter and a HeNe laser with an excitation wavelength of 633 nm; emission wavelength was optimized for each Protein Labeling • Atto 488 is a superior alternative to widely used fluorescein, dye conjugate tested. The RFI signal of the Atto dye-labeled anti- providing more photostable conjugates and brighter fluores- IgG at the emission wavelength of 650-665 nm was 6 to 13-fold cence. It can be excited with the same light sources as fluores- higher than the signals from the other dyes tested. cein dyes or Alexa Fluor 488, and the same optical filter sets and instrument settings can be used to record the emission. • For longer wavelengths, Atto 550 is efficiently excited by HeNe Dye Molar Ratio Dye/Antibody RFI* lasers and can be used as an alternative to rhodamine dyes, Cy3 or Alexa Fluor 555, offering more intense brightness and Atto 647N 4.4 130,000 increased photostability. Alexa Fluor 647 3.5 10,000 • Atto 594 is excited by HeNe lasers and can be used as alterna- Cy5 3.5 20,000 tive to Alexa Fluor 594 or Texas Red®. Table 2. * RFI values are in terms of arbitrary units. • Atto 647N, a highly fluorescent dye, and Atto 633 are both well suited for excitation with HeNe or krypton lasers, similar to Cy5 or Alexa Fluor 647. Components: The data in Tables 1 and 2 demonstrate the superior fluores- • 5 vials containing reactive Atto dye (optimized for 1 mg protein cence of Atto dye-labeled proteins. Monoclonal anti-mouse IgG sample) was labeled using Atto 550, Alexa Fluor 555, or Cy3 dyes (see • Sodium bicarbonate reaction buffer solution, pH 8.4 Table 1). The relative fluorescence intensity signals (RFI) of the dye-labeled antibody solutions were measured using a spectro- • Phosphate buffer solution, pH 7.5 fluorimeter and a HeNe laser with an excitation wavelength of • Protein purification set containing 5 spin columns, 5 washing 544 nm; emission wavelength was optimized for each dye conju- tubes and 5 sample collection tubes gate tested. The RFI signal of the Atto dye-labeled anti-IgG at the • Technical bulletin emission wavelength of 570-585 nm was 2 to 4-fold higher than the signals from the other dyes tested. Ordering Information

Recommended Excitation Cat. No. Name Wavelength 92313 Atto 488 Protein Labeling Kit 488 nm 77508 Atto 550 Protein Labeling Kit 544 nm 66314 Atto 594 Protein Labeling Kit 594 nm, 601 nm 04634 Atto 633 Protein Labeling Kit 633 nm, 647 nm 89821 Atto 647N Protein Labeling Kit 633 nm, 647 nm

For more information on Atto labels, please visit our sigma-aldrich.com/biofiles Web site at sigma.com/atto. 10 Fluorescence Multiplexing Using Antibody-Atto Dye Conjugates

Immunoblotting (Western blot transfer) is a common technique in modern proteomics research. After electrophoresis, proteins are immobilized on a nitrocellulose or PVDF membrane and then probed with a primary antibody against the protein-of-interest. A 1 + 2 secondary antibody conjugated to horseradish peroxidase or alka- Protein 1 Protein 2 line phosphatase is subsequently bound to the primary antibody. A chemiluminescent enzyme substrate is added, and the resulting light is detected using a CCD camera or X-ray film exposure. Typically, immunoblot detection targets a single protein. In certain applications, however, such as the comparison of a protein in its 100 - phosphorylated and nonphosphorylated states, or the determination of the relative abundance of a protein of interest to a control

protein (e.g., actin), multiplex detection on a single membrane is Protein 1: Atto 550 45 - desirable. Protein 2: Atto 633 Sigma offers secondary antibodies covalently linked to brightly fluorescent Atto dyes. Immunoblot multiplexing can be performed 30 - by using two secondary antibodies conjugated to different Atto dyes with different excitation/emission wavelengths (see Figure 1). Blotting membranes do not need to be stripped and reprobed for additional detection, reducing handling and minimizing sources of 12 - error.

Two-Color Multiplex Immunoblotting In our first experiment, Protein 1 and Protein 2 (500 ng each) were separated on a 4-20% SDS-PAGE (Tris-Glycine) gel. The proteins

Fluorescence Multiplexing Fluorescence were transferred to a low-fluorescence PVDF membrane and Figure 2. Immunoblot detection of Protein 1 and Protein 2 using two blocked using 5% BSA in PBS. The membrane was incubated with primary antibodies and two anti-IgG-Atto dye conjugates. Both secondary antibodies to Protein 1 and Protein 2 (1:1000 each). antibodies were developed in the same species (goat). Imaging was done sequentially using a FLA-3000 Fuji® laser scanner, first at an excitation wavelength of 532 nm with a 580 nm emission filter, then at an excitation wavelength of 633 nm with a 675 nm emission filter. The image overlay Atto Atto was done using a software tool. See text for further details.

Secondary antibody Routine Immunoblotting using Atto Dye Conjugated Antibodies Primary Atto dye conjugated antibodies can also be used for routine antibody immunoblotting. The limits of detection for amino-terminal FLAG-BAP™ fusion protein (Cat. No. P7582) using goat anti- mouse-IgG-Atto 647N (Cat. No. 50185) and goat anti-mouse- IgG-Atto 550 (Cat. No. 43394) conjugates are shown in separate Protein 1 Protein 2 experiments (see Figure 3). FLAG-BAP protein (5 - 500 ng) was separated on a 4-20% SDS-PAGE gel. The protein was transferred Figure 1. Schematic for multiplex application using two secondary anti- to low-fluorescence PVDF membranes and blocked overnight bodies with different Atto dyes. using 5% BSA in PBS. The membranes were incubated with monoclonal ANTI-FLAG® M2 antibody (Cat. No. F1804, diluted Protein 1 was probed with a primary antibody developed in 1:1000). One membrane was detected using goat anti-mouse- mouse, followed by goat anti-mouse-IgG-Atto 550 (Cat. No. IgG-Atto 647N (1:1250) (λ = 633 nm, λ = 675 nm). The mini- λ λ ex em 43394, diluted 1:1250) ( ex = 532 nm, em = 580 nm). Protein 2 mum amount of FLAG-BAP protein detected was 5 ng. A second was probed with a primary antibody developed in rabbit, followed membrane was detected using goat anti-mouse-IgG-Atto 550 by goat anti-rabbit-IgG-Atto 633 (Cat. No. 41176, diluted 1:1250) (1:1250) (λ = 532 nm, λ = 580 nm). The minimum amount of λ λ ex em ( ex = 633 nm, em = 675 nm). Both proteins are detected on a FLAG-BAP protein detected was 20 ng. For the detection, we used single membrane, when run in separate lanes and when combined a 532 nm laser combined with a 580 nm emission filter for Atto (see Figure 2). 550, and a 633 nm laser with a 675 nm emission filter for Atto 647N. Other imaging systems are possible with the corresponding excitation sources and emission filter settings. sigma-aldrich.com/biofiles 11

20 ng 500 ng 100 ng 50 ng 20 ng Marker 500 ng 50 ng 5 ng 5 ng Marker AB100 ng

100 -

60- 45 -

30 - Fluorescence Multiplexing 20-

Figure 3. Detection of FLAG-BAP protein (500 ng – 5 ng) by immunoblotting using Atto dye labeled antibodies. Image A: Antibody used was goat anti-mouse-IgG-Atto 647N. Imaging was performed at an excitation wavelength of 633 nm with a 675 nm emission filter. Image B: Antibody used was goat anti-mouse-IgG-Atto 550. Imaging was performed at an excitation wavelength of 532 nm with a 580 nm emission filter. Imaging was performed on a FLA-3000 Fuji laser scanner. See text for further experimental details.

Additional Recommendations • Incubation with the primary antibodies can be done sequentially without pooling, so that the antibodies can be reused. • In order to avoid cross-reactivity, primary antibodies must be derived from different hosts, and secondary antibodies must be derived from the same host. • A low-fluorescence PVDF membrane such as ImmobilonTM-FL must be used to avoid high background. • Drying the membranes enhances the signal intensity.

Atto-labeled antibodies offer new possibilities for immunoblot detection without enzymatic substrates as well as for single membrane multiplexing applications. Ordering Information

Cat. No. Name Developed In Recommended lex Maximum lem Pack Size 62197 Anti-Mouse IgG - Atto 488 Goat 488 nm 521 nm 1 mL 43394 Anti-Mouse IgG - Atto 550 Goat 543 nm 574 nm 1 mL 76085 Anti-Mouse IgG - Atto 594 Goat 594 nm, 601 nm 627 nm 1 mL 78102 Anti-Mouse IgG - Atto 633 Goat 633 nm 650 nm 1 mL 50185 Anti-Mouse IgG - Atto 647N Goat 633 nm, 647 nm 667 nm 1 mL 50283 Anti-Mouse IgG - Atto 655 Goat 655 nm 680 nm 1 mL 12708 Anti-Mouse IgG - Mega 485 Goat 485 nm 559 nm 1 mL 39304 Anti-Mouse IgG - Mega 520 Goat 520 nm 667 nm 1 mL 18772 Anti-Rabbit IgG - Atto 488 Goat 488 nm 521 nm 1 mL 36098 Anti-Rabbit IgG (Fab specific) F(ab´)2 fragment - Atto 488 Goat 488 nm 521 nm 1 mL 43328 Anti-Rabbit IgG - Atto 550 Goat 544 nm 574 nm 1 mL 68919 Anti-Rabbit IgG - Atto 590 Goat 590 nm 620 nm 1 mL 77671 Anti-Rabbit IgG - Atto 594 Goat 594 nm, 601 nm 627 nm 1 mL 41176 Anti-Rabbit IgG - Atto 633 Goat 633 nm 650 nm 1 mL 40839 Anti-Rabbit IgG - Atto 647N Goat 633 nm, 647 nm 667 nm 1 mL 78519 Anti-Rabbit IgG - Atto 655 Goat 655 nm 680 nm 1 mL 38376 Anti-Rabbit IgG - Mega 520 Goat 520 nm 667 nm 1 mL

02295 Anti-Rabbit IgG - Mega 485 Goat 485 nm 559 nm 1 mL sigma-aldrich.com/biofiles 50913 Anti-Chicken IgG - Atto 590 Rabbit 590 nm 620 nm 1 mL

Related Products Cat. No. Name Pack Size 05317 ImmobilonTM-FL PVDF Membrane 10 ea

For more information on Atto Dye Conjugates, visit our Web site at sigma.com/conjugates. 12 Ni-NTA-Atto Conjugates

Ni-NTA-Atto Conjugates for Sensitive, Speciﬁ c Direct Detection of Histidine-Tagged Proteins on Detection of Polyhistidine-Tagged Proteins SDS-PAGE Gels Direct application of Ni-NTA-Atto conjugates to SDS-PAGE gels State-of-the-art recombinant protein applications require reliable provides fast and easy detection for monitoring protein purifica- methods of detection. Polyhistidine is one of the most popular tion or protein expression at different points of time. A detection affinity tags incorporated into recombinant proteins. It can be limit of 50 ng for His-tagged p38 MAPK was observed using fluo- inserted either at the N- or C-terminus, and expressed in a vari- rescence imaging (see Figure 2). ety of hosts. Due to its small size, the polyhistidine tag serves as an elegant tool for both protein purification and detection. Immunodetection of His-tagged fusion proteins on a Western blot 25 ng 250 ng 100 ng 500 ng Marker typically uses an antibody to polyhistidine, followed by a second- 50 ng ary antibody conjugated to an enzyme such as horseradish peroxidase or alkaline phosphatase. The protein is then detected with an appropriate enzyme substrate. 75- Ni-NTA-Atto conjugates provide specific and highly sensitive detec- 50- tion of His-tagged fusion proteins. The Ni-NTA-Atto complex p38 MAPK (Nα,Nα-bis(carboxymethyl)-L-lysine, Nickel(II) complex, conjugated to Atto dye) is specific for polyhistidine tags with minimal cross- 30- reactivity. Atto dyes deliver strong fluorescent signals at commonly available wavelengths and with little quenching. Ni-NTA-Atto conjugates can be directly applied either to an SDS-PAGE gel or Western blot membrane for fluorescence imaging, and have been successfully used in living cells.1,2 Detection with Ni-NTA-Atto conjugates requires less incubation time than for protein-anti- Figure 2. His-tagged p38 MAPK protein (500 ng – 25 ng) was separated body binding. No secondary reaction is required, since the Ni-NTA on a 4-20% Tris-Glycine SDS-PAGE gel. The gel was fixed overnight in 40%

Ni-NTA-Atto Conjugates Ni-NTA-Atto complex is directly conjugated to the fluorophore. This results in a ethanol/10% acetic acid, washed in water and incubated with Ni-NTA-Atto 647N (1:1000) in the dark. The gel was washed and then imaged using a faster and more flexible procedure than the traditional immunode- FLA-3000 Fuji® laser scanner with 633 nm excitation and a 675 nm emis- tection method (see Figure 1). sion filter. Ni-NTA-Atto 647N (lex 647 nm, lem 669 nm) is excited in the red region of the spectrum.

CLCL Chemiluminescent detection Detection of Histidine-Tagged Proteins after Western Blot Transfer Secondary After electrophoresis, proteins may be transferred from SDS-PAGE Antibody gels to low-fluorescence PVDF-membranes, which are more robust AttoAtto to handling. Traditional Western blots work well for polyhistidine Antibody to tags, but are time consuming. Ni-NTA-Atto conjugates combine polyhistidine Ni-NTA the advantages of highly specific detection with a more rapid procedure. His His Application of Ni-NTA-Atto conjugates to the membrane yields sensitivity comparable to direct gel detection. Drying the mem- Protein Protein brane enhances the signal intensity (see Figure 3). In addition, the Ni-NTA-Atto conjugates may be stripped from the used membrane Traditional Ni-NTA-Atto conjugate with a 60-90 minute incubation in 20 mM EDTA. Figure 1. Comparison of protein detection by traditional antibody chemiluminescent immunodetection (left) to Ni-NTA-Atto conjugate detection (right). Immunodetection takes place on a transfer membrane after Western blotting, while the Ni-NTA-Atto conjugates may be used with either transfer Marker 25 ng 500 ng 250 ng 50 ng membranes or directly on SDS-PAGE gels after fixing. 100 ng

75-

50- p38 MAPK

30-

Figure 3. His-tagged p38 MAPK protein (500 ng – 25 ng) was separated on a 4-20% SDS-PAGE gel. The protein was transferred to a low-fluorescence PVDF-membrane, blocked overnight with 5% BSA in PBS, rinsed with PBS-T, and incubated with Ni-NTA-Atto 647N (1:1000) in the dark. The membrane was washed and then imaged using a FLA-3000 Fuji laser scanner with 633 nm excitation and a 675 nm emission filter. sigma-aldrich.com/biofiles 13

Traditional Immunoblotting Western Blot using Direct Detection using Procedure NI-NTA-Atto conjugate Ni-NTA-Atto conjugate

SDS-PAGE SDS-PAGE SDS-PAGE Membrane transter (1-2 hr) Gel fixation in 40% ethanol: 10% acetic acid Membrane transfer (1-14 hr [overnight]) (1-2 hr) Blocking with 5% BSA in PBS (1-14 hr [overnight]) Rinse with PBST Blocking with 5% BSA in PBS Ni-NTA-Atto Conjugates (1-14 hr [overnight]) Wash with water (2330 min) Incubate with primary antibody Rinse with PBST (2-3 hr) Wash with PBST Incubate with Ni-NTA-Atto Incubate with Ni-NTA-Atto (335 min) 1:1000-1:2500 in PBST 1:1000 - 1:2500 in PBST (1hr, dark) (1 hr, dark) Inclubate with secondary Wash with PBST antibody (1 hr) Wash with water (0-1hr dark) (1-2 hr, dark) Wash with PBST (335 min) Fluorescent imaging Chemiluminescent detection Fluorescent imaging

Total Time 5.5-20.5 hours Total Time 3-18 hours Total Time 4-18 hours

Number of Steps 8 Number of Steps 6 Number of Steps 5

Table 1. Comparison of the protocols for traditional Western immunoblot, Western blot using Ni-NTA-Atto conjugate, and direct detection on an SDS-PAGE gel using Ni-NTA-Atto conjugate. Ni-NTA-Atto stock solution is prepared by dissolving 250 µg in 250 µL PBS.

Comparison of procedures for the traditional Western immunoblot Ordering Information and Ni-NTA-Atto conjugates is shown in Table 1. Direct SDS-PAGE gel detection is the most convenient as it omits the transfer step. Cat. No. Name Pack Size Similar detection limits with Ni-NTA-Atto conjugates are observed 02175 NTA-Atto 647N 250 µg for Western blot membranes compared to SDS-PAGE gels, but 94159 NTA-Atto 550 250 µg without risk of gel damage. In either case, the hands-on time is less than for the traditional antibody technique. 39625 NTA-Atto 488 250 µg Ni-NTA-Atto conjugates provide a valuable alternative to traditional antibody immunodetection for specific and sensitive detection of His-tagged fusion proteins. Use of Ni-NTA-Atto reduces Related Products experiment time and eliminates time-consuming antibody valida- Cat. No. Name Pack Size tion experiments, saving both time and expense. 05317 Immobilon-FL PVDF membrane 10 ea References: 1. Single Molecule Detection with Atto 647N NTA, BioFiles, Issue 3, pp. 8-9, Sigma-Aldrich (2006). For additional information on related products 2. Guignet, E.G., et al., Reversible site-selective labeling of membrane for histidine-tagged proteins, visit our Web site at proteins in live cells. Nat. Biotechnol., 22, 440-4 (2004). sigma.com/hisselect. sigma-aldrich.com/biofiles 14 Fluorogenic Substrates

Fluorogenic Substrates for the Sensitive Sigma has a broad offering of fluorogenic enzyme substrates that Detection of Enzymes selectively release the fluorescent moiety after the cleavage by the enzyme of interest. Fluorescent detection is used for elucidation of High specificity and ultra-sensitive measurement of enzymatic enzyme function in a variety of qualitative and quantitative appli- activity is becoming increasingly important in life science research. cations, including: Many of the traditional high sensitivity methods such as detection of radioactive end-products released from isotopically-labeled substrates require specialized instrumentation, disposal issues, and • Screening potential enzyme inhibitors a high degree of safety and regulatory oversight. Simple, robust • Evaluation of proteins for enzymatic activity via directed evolu- assay procedures are needed which can be routinely performed tion and site-directed mutagenesis in all laboratories using commonly available scientific instruments. • Identification and quantitation of trace enzymatic activity in Straightforward and inexpensive detection using fluorescence has natural source products and metagenomes tremendous advantages, including high specificity, substrate stability, and a reliable reagent supply chain. Today’s fluorescent • Analysis of residual enzyme impurities in enzyme preparations techniques are yielding limits of detection comparable to radioactive techniques.

Fluorogenic Substrates

Enzyme Fluorogenic Substrate lex (nm) lem (nm) Cat. No. Pack Size

β-N-Acetyl-D-galactosaminidase 4-Methylumbelliferyl N-acetyl-β−D-galactos- 362 448 M9659 25 mg aminide 100 mg 250 mg 1 g Fluorogenic Substrates Fluorogenic β-N-Acetyl-D-glucosaminidase 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide 362 448 M2133 25 mg 100 mg 250 mg 1 g Alkaline Phosphatase 4-Methylumbelliferyl phosphate 364 448 M8883 100 mg 250 mg 1 g 5 g Alkaline Phosphatase 4-Methylumbelliferyl phosphate 360 440 M3168 100 mL Liquid Substrate System Alkaline Phosphatase 4-Methylumbelliferyl phosphate disodium salt 364 448 M8168 250 mg 1 g

Aminopeptidase L-Alanine 7-amido-4-methylcoumarin 325 389 A4302 25 mg trifluoroacetate salt 100 mg 250 mg

Aminopeptidase Z-L-Arginine 7-amido-4-methylcoumarin 290 390 C8022 50 mg hydrochloride

Aminopeptidase Z-Glycyl-L-proline 7-amido-4-methylcoumarin 290 390 96280 25 mg 100 mg

Aminopeptidase B L-Leucine 7-amido-4-methylcoumarin 325 397 61888 25 mg hydrochloride

Aminopeptidase M L-Phenylalanine 7-amido-4-methylcoumarin 320 383 78089 25 mg trifluoroacetate salt 100 mg

α-L-Arabinofuranosidase 4-Methylumbelliferyl α-L-arabinofuranoside 364 448 M9519 10 mg 50 mg 100 mg Butyrate esterase 4-Methylumbelliferyl butyrate 365 445 19362 1 g 5 g Note: Excitation and emission wavelength information has been experimentally determined or is taken from scientific literature. sigma-aldrich.com/biofiles 15

Fluorogenic Substrates

Enzyme Fluorogenic Substrate lex (nm) lem (nm) Cat. No. Pack Size

Cellulase 4-Methylumbelliferyl β-D-cellobioside 365 455 M6018 100 mg 500 mg

Chitinase 4-Methylumbelliferyl N,N’-diacetyl-β-D-chitobio- 330 375 M9763 5 mg side hydrate 10 mg 25 mg

α-Chymotrypsin N-(N-Glutaryl-L-phenylalanyl)-2-aminoacridone 398 440 49742 25 mg 100 mg

α-Chymotrypsin N-(N-Succinyl-L-phenylalanyl)-2-aminoacridone 398 440 85992 25 mg 100 mg Fluorogenic Substrates α-Chymotrypsin 4-Methylumbelliferyl p-trimethylammonio- 365 410 M4507 25 mg cinnamate chloride 100 mg α-Chymotrypsin N-Succinyl-Ala-Ala-Pro-Phe 7-amido-4-methyl- 380 460 S9761 1 mg coumarin 5 mg α-Chymotrypsin Ala-Ala-Phe 7-amido-4-methylcoumarin n/a n/a A3401 25 mg Collagenase Collagen Type I−FITC conjugate from bovine skin 490 520 C4361 10 mL Collagenase Collagen-Fluorescein bovine 490 520 C5858 25 mg Cytochrome P450 2B6 7-Ethoxycoumarin 323 386 E1379 25 mg 100 mg 500 mg 1 g Cytochrome P450 Resorufin benzyl ether 571 585 B1532 5 mg 10 mg Cytochrome P450 Resorufin ethyl ether 571 585 E3763 1 mg 5 mg Cytochrome P450 Resorufin methyl ether 571 585 M1544 1 mg 5 mg Cytosolic aldehyde hydrogenase Resorufin acetate 500 593 83636 25 mg Dealkylase Resorufin pentyl ester 571 585 77105 5 mg Esterase 3-(2-Benzoxazolyl)umbelliferyl acetate 410 468 12832 25 mg 100 mg Esterase 8-Butyryloxypyrene-1,3,6-trisulfonic acid 460 510 20800 25 mg trisodium salt Esterase 2’,7’-Dichlorofluorescein diacetate 504 529 D6883 50 mg 250 mg Esterase Fluorescein dilaurate 490 514 46943 1 g 5 g Esterase 4-Methylumbelliferyl acetate 365 445 M0883 5 g Esterase 4-Methylumbelliferyl butyrate 365 445 19362 1 g 5 g Esterase 8-Octanoyloxypyrene-1,3,6-trisulfonic acid 460 510 74875 25 mg sigma-aldrich.com/biofiles trisodium salt 100 mg Factor X activated (Xa) 4-Methylumbelliferyl 4-guanidinobenzoate 365 445 51010 250 mg hydrochloride hydrate 1 g 16 Fluorogenic Substrates

Fluorogenic Substrates

Enzyme Fluorogenic Substrate lex (nm) lem (nm) Cat. No. Pack Size

α-L-Fucosidase 4-Methylumbelliferyl α-L-fucopyranoside 365 445 M8527 1 mg 5 mg 10 mg 25 mg 100 mg

α-Galactosidase 4-Methylumbelliferyl α-D-galactopyranoside 365 445 M7633 25 mg 100 mg 250 mg

β-Galactosidase Fluorescein-di(β-D-galactopyranoside) 490 514 F2756 1 mg 5 mg 25 mg

β-Galactosidase 4-Methylumbelliferyl β-D-lactopyranoside 365 445 M2405 25 mg 100 mg

β-Galactosidase Resorufin β-D-galactopyranoside 571 590 R4883 10 mg 100 mg

β-Glucosidase 4-Methylumbelliferyl β-D-glucopyranoside 365 445 M3633 100 mg 250 mg 500 mg 1 g Fluorogenic Substrates Fluorogenic 5 g

β-Glucosidase Resorufin β-D-glucopyranoside 571 590 R4758 10 mg 50 mg β-Glucosidase 4-(Trifluoromethyl)umbelliferyl 385 502 91880 25 mg β-D-glucopyranoside 100 mg β-Glucuronidase 6,8-Difluoro-4-methylumbelliferyl 366 448 36939 5 mg β-D-glucuronide lithium salt

β-Glucuronidase 4-Methylumbelliferyl β-D-glucuronide hydrate 365 445 69602 250 mg 1 g

Leucine Aminopeptidase L-Leucine 7-amido-4-methylcoumarin 325 397 61888 25 mg hydrochloride Lipase Fluorescein dilaurate 490 514 46943 1 g 5 g Lipase 4-Methylumbelliferyl butyrate 365 445 19362 1 g 5 g Lipase 4-Methylumbelliferyl heptanoate 365 445 M2514 100 mg 250 mg 1 g Lipase 4-Methylumbelliferyl oleate 327 449 75164 25 mg 100 mg Lipase 4-Methylumbelliferyl palmitate 370 450 M7259 100 mg Lipase 4-Methylumbelliferyl stearate 370 450 M1010 100 mg Lipase Resorufin butyrate 500 593 83637 25 mg 100 mg

Lysozyme 4-Methylumbelliferyl β-D-N,N’,N’’-triacetylchito- 360 455 M5639 1 mg trioside 5 mg

a-Mannosidase 4-Methylumbelliferyl α-D-mannopyranoside 347 435 M3657 10 mg 25 mg 100 mg sigma-aldrich.com/biofiles 17

Fluorogenic Substrates

Enzyme Fluorogenic Substrate lex (nm) lem (nm) Cat. No. Pack Size b-Mannosidase 4-Methylumbelliferyl β-D-mannopyranoside 355 460 M0905 10 mg 25 mg 100 mg 250 mg Monooxygenase 7-Ethoxycoumarin 323 386 E1379 25 mg 100 mg 500 mg 1 g Fluorogenic Substrates Neuraminidase 2’-(4-Methylumbelliferyl) α-D-N-acetyl-neuramin- 365 445 69587 5 mg ic acid sodium salt hydrate 25 mg

Papain Z-L-Arginine 7-amido-4-methylcoumarin 290 390 C8022 50 mg hydrochloride Peroxidase Ampliflu™ Red 571 585 90101 5 mg Peroxidase Dihydrorhodamine 123 500 525 D1054 2 mg 10 mg Phosphodiesterase 1-Naphthyl-4-phenylazophenyl phosphate n/a n/a 10922 250 mg Phosphodiesterase 2-Naphthyl-4-phenylazophenyl phosphate n/a n/a 10923 50 mg 500 mg Sulfatase 4-Methylumbelliferyl sulfate potassium salt 365 445 M7133 500 mg 1 g Thrombin 4-Methylumbelliferyl 4-guanidinobenzoate 365 445 51010 250 mg hydrochloride hydrate 1 g

Trypsin Z- L-Arginine 7-amido-4-methylcoumarin 290 390 C8022 50 mg hydrochloride

β-Xylosidase 4-Methylumbelliferyl β-D-xylopyranoside 365 445 M7008 25 mg 100 mg 250 mg 1 g

The New Enzyme Explorer • Redesigned homepage user interface

• Expanded and reformatted sigma-aldrich.com/biofiles technical resources and indexes • Protease Finder: Identifies the cleavage sequence sigma.com/enzymeexplorer 18 Ampliﬂ u™ Red Peroxidase Substrate

Ampliﬂ u Red, a Fluorescent Peroxidase Substrate for Immunoblot Applications 40 ng 50 ng 30 ng 20 ng 10 ng 5 ng Marker 1 ng Protein detection by immunoblotting often incorporates horseradish peroxidase (HRP) labeled secondary antibodies. Peroxidase can be detected directly using colorimetric substrates such as 3,3’,5,5’- tetramethylbenzidine (TMB) and 3,3’-diaminobenzidine (DAB), or by using chemiluminescence-based substrates with light emission measured using a CCD camera (charged coupled device) or X-ray film. Chemiluminescent methods are usually more sensitive than those employing colorimetric substrates. FLAG-BAP Ampliflu Red is a peroxidase substrate that is enzymatically converted to the highly fluorescent compound resorufin. Fluorescent imaging is performed on the immunoblot membrane using a laser scanner with an excitation wavelength of 571 nm and an emission filter of 585 nm. Other imaging systems with a similar excitation source and emission filter settings may also be used. Ampliflu Red

Substrate can detect 1 ng of protein with just a simple fluorescence readout. There is no prolonged signal accumulation time as required for Figure 2. Detection of FLAG-BAP protein (50–1 ng) by immunoblotting using Ampliflu Red (1880 µl PBS / 20 µl Ampliflu Red from 10 mM stock/ chemiluminescent measurements. 100 µL H2O2 from 20 mM stock). Imaging was performed on a FLA-3000 Fluorescence Fuji laser scanner at a fixed excitation wavelength of 532 nm with a Excitation Imaging 580 nm emission filter. See text for further experimental details. Ampliflu Red is a reliable new chemifluorescent substrate for the specific and sensitive detection of peroxidase labeled antibodies on Western blots. It is a convenient alternative to conventional Ampliflu Red Peroxidase Ampliflu Red Peroxidase colorimetric or chemiluminescent immunoblotting protocols and Ampliflu Red Resorufin has the advantage of generating a high signal-to-noise ratio, without the need for long signal accumulation times. Depending on the available instrumentation, Ampliflu Red can be measured on Secondary antibody - HRP a laser scanner or similar instruments capable of fluorescent measurements of membranes. Primary antibody Ordering Information Cat. No. Name Pack Size Protein Membrane 90101 Ampliflu Red 5 mg

05317 Immobilon-FL PVDF membrane 10 ea Figure 1. Schematic overview for the use of Ampliflu Red. A protein is immobilized by Western blot transfer after SDS-PAGE. The protein is bound with a primary antibody and followed by a secondary antibody carrying a horseradish peroxidase (HRP) label. The membrane is incubated in a solution containing Ampliflu Red and hydrogen peroxide. Ampliflu Red is converted into resorufin by the action of HRP. The resultant fluorescent signal can be detected at the excitation and emission parameters of resorufin λ λ ( ex = 571 nm, em = 585 nm).

Amino-terminal FLAG-BAP Fusion Protein (Cat. No. P7582, 1–50 ng) was separated on a 4-20% SDS-PAGE gel and transferred to a low-fluorescence Immobilon™-FL PVDF membrane (Cat. No. 05317). This membrane is recommended because it optimizes the signal-to-noise ratio. The membrane was blocked using 5% BSA in PBS and then incubated with monoclonal ANTI- FLAG M2 antibody developed in mouse (Cat. No. F1804, diluted 1:1000). The secondary antibody used was anti-mouse-IgG-peroxidase developed in rabbit (Cat. No. A9044, diluted 1:10,000); the membrane was then washed and incubated with a solution of Ampliflu Red (100 µM) and hydrogen peroxide (1 mM) in PBS (2 ml total volume). After a 5 minute incubation, imaging was performed at an excitation wavelength of 532 nm with a 580 nm emission filter, resulting in an image with low background. sigma-aldrich.com/biofiles 19 Certiﬁ ed Fluorescence Standards

U. Resch-Genger, D. Pfeiffer, A. Hoffmann, Federal Institute for The ability to compare fluorescence data and the use of fluores- Materials Research and Testing (BAM), Germany cence spectra for the identification of analytes rely on the knowl- λ R. Meier, A. Rück, P. Nording, B. Schönenberger Sigma-Aldrich, edge and consideration of s( em) and its time-dependent changes Buchs, Switzerland due to degradation of the instrument components.7 The latter λ makes regular control of s( em) mandatory but simultaneously comprises an elegant tool for IPV and control of instrument long- There is an increasing need for reliable and comparable data, term stability (see Figure 2). quantification, and standardization for luminescence techniques that are widely used in the life sciences, material sciences and 1011 1-4

environmental analysis. Instrumentation can be a major source -1 1010 of variability, making comparison of fluorescence measurements sr -3 tungsten ribbon lamp between instruments and over time difficult. Standardization can 109 sphere type radiator be achieved with reliable standards in combination with specific 108 fluorescence spectra of protocols for instrument characterization and instrument perfor- selected dyes Standards Certified Fluorescence mance validation (IPV).5 107

With assistance from Sigma, the Federal Institute for Materials 106 Research and Testing (BAM, Berlin, Germany) has developed the 5 Spectral Fluorescence Standards Kit.6 10 104

The Spectral Fluorescence Standards Kit provides a simple and spectral radiance /Wm flexible calibration tool for the determination and control of the 103 relative spectral responsiveness of fluorescence instruments. The 300 400 500 600 700 kit is optimized for use with spectrofluorometers, provides trace- wavelength (nm) ability of fluorescence measurements to the spectral radiance Figure 2. Comparison of the typical spectral radiance of a tungsten ribbon scale, and can be used with various measurement geometries. It lamp (top, red), an integrating sphere type radiator (middle, green), and can be adapted for calibration of other fluorescence measuring typical fluorophores (bottom, blue). instruments with proper consideration of the underlying measurement principles Determination of Spectral Responsivity The spectral responsivity value of an instrument can be obtained Instrument Effects on Fluorescence Signals by evaluating a reference material that emits a known and broad Fluorescent signals are influenced by both sample- and instrument spectrum in the ultraviolet/visible/near infrared region.7,8 In general, three primary types of light sources can be used: specific effects. In practice, it is difficult to reproduce fluorescent data on different instruments or at different times with the same • Lamps (e.g. tungsten ribbon lamps) instrument, regardless of experimental parameters. This variance in results is due to instrument-specific contributions to the fluores- • Sphere-type spectral radiators cent signal. These wavelength-, polarization- and time-dependent • Fluorophore standards differences reflect:

Tungsten ribbon lamps and integrating sphere-type spectral radi- • The spectral radiance of the excitation light source ance transfer-standards are traceable, but are tedious to align, • The transmittance of optical components, including lenses, can impose restrictions on measurement geometry, require regular mirrors, filters, monochromator gratings, and polarizers in the calibration, which can be expensive, and often do not physically fit excitation and emission channels into compact fluorescence instruments.7,8 Moreover, their spectral • The spectral responsivity of the detection system radiances/emission intensities exceed those of typical fluorescent samples by approximately two and four orders of magnitude, respectively, for integrating sphere radiators and tungsten ribbon The influence of the spectral responsivity s(λ ) on fluorescence em lamps (see Figure 2). Accordingly, to avoid errors due to the non- data and the resultant need for its consideration, i.e., spectral linearity of the detection system, their use requires attenuation emission correction, are illustrated for a typical organic fluoro- procedures that do not introduce additional spectral effects.6 phore analyzed using two spectrofluorometers (see Figure 1). In contrast, fluorophore-based spectral fluorescence standards7,9-13 are easy to use and offer flexibility with respect to measurement geometry and instrument type. These standards can be measured under routine conditions, reducing many sources of systematic error. Fluorophore standards must be selected to encompass the ultraviolet/visible/near infrared spectral region. Each standard must also give a broad and unstructured emission spectrum to mini- sigma-aldrich.com/biofiles mize the influence of spectral bandwidth on spectrum shape, and have a very small fluorescence anisotropy to minimize polarization effects and ameliorate the need for polarizers.6,7,14

norm. fluorescence Additional requirements for spectral fluorescence standards are: (1) Traceable and accurate measurement of their corrected emis- 550 600650 700 750 sion spectra with a reported uncertainty. wavelength (nm) (2) Characterization of their calibration-relevant properties Figure 1. Normalized uncorrected (open symbols, blue and green) and according to EN ISO/IEC 17025 and ISO Guides 34 and 35. corrected (full symbols) emission spectra of a typical organic fluorophore When properly selected, these standards can be measured under measured with two different fluorometers. routine conditions, reducing sources of systematic error and providing the basis for comparable emission data. 20 Certiﬁ ed Fluorescence Standards

Principle and Properties Traceability, Certiﬁ cation and Proﬁ ciency Testing The Spectral Fluorescence Standards Kit consists of: The Spectral Fluorescence Standards Kit has been certified by

BAM according to ISO Guidelines 34 and 35, and the standards • Five spectral fluorescence standards covering the spectral region are traceable to the spectral radiance scale.6-8,14 The wavelength- of 300 to 760 nm dependent relative uncertainties of the corrected emission spectra • BAM-certified normalized corrected emission spectra are a combination of the relative uncertainties of the calibration • Ethanol for use as a solvent of the fluorometer used for certification, the measurements of the • LINKCORR software. The LINKCORR software was developed emission spectra, and material-related uncertainties derived from λ homogeneity and stability studies.14 and evaluated by BAM for the calculation of s( em) based on the measured and certified emission spectra of the standards pro- The corrected emission spectra of the kit standards and the vided in the kit. determination of an emission correction curve using the Spectral • Technical insert with calibration procedure Fluorescence Standards Kit have been evaluated by sequential analysis performed by: The emission spectra, LINKCORR software, and technical insert are • The National Metrological Institutes (NIST, U.S.A.) provided on a single compact data disk. • The National Research Council of Canada (NRC) The five standards have been selected to match a set of spectral • The Physikalische Technische Bundesanstalt (PTB, Germany) properties requirements.13,14 The standards are also available sepa- Standards rately. Working solutions of each of the fluorescent standards are • Federal Institute for Materials Research and Testing measured with the instrument to be calibrated, and data is evalu- (BAM, Berlin, Germany) ated with the provided LINKCORR software (see Figure 3). The laboratories used four characterized instruments and two B different measurement geometries (0°/90° and 45°/0°). In addition, the Spectral Fluorescence Standards Kit has been successfully tested with several types of common spectrofluorometers.7,14 The Certified Fluorescence Certified Fluorescence FOOX results depicted in Figure 4 compare the uncorrected and standard-corrected emission spectra of three test dyes X, QS, and Y. The corrected fluorescence spectra demonstrate a variance of less than 10%. This precision is similar to what is achievable by expe- Quotient Q rienced technicians using physical transfer standards, tedious and time-consuming procedures, and severe geometry restrictions.7,14

A A 1.0 Uncorrected 0.8 spectra 0.6 0.4 X QS Y

Fluorescence 0.2

norm. fluorescence 0.0 300 350 400 450 500 550 600 650 700 nm

300 400 500 600 700 nm B 1.0 wavelength(l ) em Corrected 0.8 spectra A ------Iu (lem) — F001 for each of the standards F001-F005 — F002 0.6 ——– I (l ) — F003 c em 0.4 for each of the standards F001-F005 — F004 B — F005 0.2 Relative spectral responsivity (S(l))

——– norm. fluorescence 0.0 Combined correction curve (I/S(l)) 300 350 400 450 500 550 600 650 700 nm

wavelength(lem) Figure 3. Working principle of the Spectral Fluorescence Standards Kit. See text for detailed explanation. Figure 4. Proficiency testing of the Spectral Fluorescence Standards Kit. Kit-based spectral correction of A) the uncorrected emission spectra λ of the test dyes X, QS, and Y measured with four fluorometers and The LINKCORR software calculates the quotients QF00x( em) of λ B) comparison of the resulting corrected emission spectra. A variance of the certified emission spectra Ic( em) (see Figure 3A, solid lines) λ <10% is achieved after correction. and the uncorrected emission spectra Iu( em) (Figure 3B, dashed lines) for each dye. As determined by the LINKCORR software, a λ weighted combination of QF00x( em) (Figure 3B, solid color lines) yields an overall emission correction curve (Figure 3B, solid black line). This curve equals the inverse relative spectral responsivity λ of the instrument (1/s( em)). The relative spectral responsivity of the instrument (S(λ)) is indicated in Figure 3B (broken black line). Corrected instrument-independent and comparable data are then obtained upon multiplication of measured spectra with this correc- λ tion curve (1/s( em)). sigma-aldrich.com/biofiles 21

By combining multiple standards, convenient software analysis, 6. Resch-Genger, U., et al., Traceability in fluorometry: Part II. Spectral and independent validation, the Spectral Fluorescence Standards fluorescence standards, J. Fluoresc., 15, 315-336 (2005). Kit now allows researchers using fluorescent detection to establish 7. Monte, C., Linking fluorescence measurements to radiometric units, Metrlogie., 43, S89-S93 (2006). comparable and traceable fluorescent measurements and to iden- 8. Hollandt, J., et al., Traceability in fluorometry: Part I. Physical transfer tify valid instrument performance. standards, J. Fluoresc., 15, 301 - 313 and references therein (2005). 9. Miller, J.N., Standards in Fluorescence Spectrometry, Ultraviolet Ordering Information Spectrometry Group, London (1981). 10. Velapoldi, R.A. and Tonnesen, H.H., Corrected emission spectra and Cat. No. Name Pack Size quantum yields for a series of fluorescent compounds in the visible spectral 97003 Spectral Fluorescence Standards Kit 1 kit region, J. Fluoresc., 14, 465-472 (2004). 72594 Fluorescence Spectral Standard BAM-F001 1 vial 11. Hofstraat, J.W. and Latuhihin, M.J., Correction of fluorescence-spectra, Appl. Spectr., 48, 436-447 (1994). 23923 Fluorescence Spectral Standard BAM-F002 1 vial 12. Velapoldi, R.A. and Mielenz, K.D., A fluorescence standard reference

96158 Fluorescence Spectral Standard BAM-F003 1 vial material: Quinine sulfate dihydrate, NBS Spec. Publ. 260-64, PB 80132046, Standards Certified Fluorescence 74245 Fluorescence Spectral Standard BAM-F004 1 vial Springfield, VA (1980). 13. Gardecki , J.A. and Maroncelli, M., Set of secondary emission 94053 Fluorescence Spectral Standard BAM-F005 1 vial standards for calibration of the spectral responsivity in emission spectroscopy, Appl. Spectr., 52, 1179-1189 (1998). References: 14. Resch-Genger, U. et al., The calibration Kit Spectral Fluorescence 1. Lakowicz, J.R., Principles of Fluorescence Spectroscopy, 2nd ed. Kluwer Standards-A simple and certified tool for the standardization of the Academic/Plenum Press, New York (1999). spectral characteristics of fluorescence instruments, J. Fluoresc., 16, 2. Lakowicz, J.R. (Ed.), Topics in Fluorescence Spectroscopy Series Vol. 1–8, 581-587, (2006). Plenum, New York (1992-2004). 15. Hoffmann, K., et al., Standards in Fluorescence Spectroscopy: 3. Wolfbeis, O.S. (Series Ed.), Springer Series on Fluorescence, Methods and Simple Tools for the Characterization of Fluorescence Instruments, G.I.T. Applications Vol. 1–3, Springer, Berlin (2001-2004). Laboratory Journal, 5, 2-4 (2005). 4. Schulman, S.G. (Ed.), Molecular Luminescence Spectroscopy Parts 1–3, Wiley Interscience, New York (1985-1993). 5. Resch-Genger, U., et al., How to improve quality assurance in fluorometry: Fluorescence-inherent sources of error and suited fluorescence standards, J. Fluoresc., 15, 337-362 and references therein (2005).

For the BAM Standard Operating Procedure and Certificate for the use of Certified Fluorescence Standards, please visit our Web site at sigma.com/fluorescence.

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Introducing the New BioUltra Proteins – risk-free, high-purity proteins, including Proteinase K, Alkaline sigma-aldrich.com/biofiles Phosphatase and Leupeptin To reserve your copy, visit us online at sigma.com/sigmacat1 22 PKH Linker Kits

PKH Linker Kits for Fluorescent Cell Labeling The PKH Fluorescent Cell Linker Kits avoid all the pitfalls of these traditional methods. Now, there is a cell labeling method that is Features the single “best way” to consistently and reliably label viable cells (see Table 1). Named for their discoverer, Paul Karl Horan, PKH • Achieve stable, uniform, intense, and reproducible dyes are patented fluorescent dyes and a cell labeling technology2 fluorescent labeling of live cells that has been used successfully with animal, plant, and bacte- • Stable labeling for up to 100 days rial cells, and even non-cellular membrane-containing particles.3 • Non-cytotoxic with no effect on biological Labeled cells can be studied in culture and in vivo. Rapidly divid- or proliferative activity ing cells like hybridomas, as well as non-proliferative cells like red • Intense and easy to use blood cells, can be labeled and tracked. • Versatile – use with any cell type Versatility • Minimal leaking or transfer from cell to cell The versatility of PKH cell-labeling technology is attributable to Introduction its innovative chemistry. Intensely fluorescent dye moieties are attached to long, lipophilic tails. During the short general mem- Many key research findings in cell function and pathogenesis brane staining procedure – only 1-5 minutes – the lipophilic tails have relied on labeling live cells. To study individual cell behavior, diffuse into the cell membrane, leaving the fluorogenic moiety growth and differentiation, and cell-cell interactions, it is critical to exposed near the outer surface of the cell. be able to permanently “tag” a population of cells without affect- ing their morphology or function. Studies of cellular phenomena Stability like adhesion, conjugate formation, cytotoxicity, phagocytosis,

PKH Linker Kits growth promotion, survival time, apoptosis, and hybridoma fusion The stable partitioning of these molecules into the membrane per- all rely on dependable nontoxic, stable cell labeling. mits long-term monitoring while leaving the important functional surface proteins unaltered. In fact, all cellular functions, from Early methods for labeling cells employed fluorescent labels such proliferation to cell-surface antigen recognition, are largely unaf- as fluorescein, rhodamine, Dil-C18-(3), or Hoechst dyes; or radio- fected by PKH dye tagging. In short, labeled cells behave just like 125 111 51 labels like I-idoxuridine, In-indium oxine and Cr-sodium unlabeled ones, but are easier to track. chromate. These methods served well in their time, but each has limitations and drawbacks.1 Some are not stable enough, so they leak and transfer to other cells in a mixed population, confound- ing results. Others alter cell function or are cytotoxic, and thus cause experimental artifacts related directly to their use. Finally, these methods often label weakly or non-uniformly, making detection difficult and design of reliable labeling protocols impossible.

Lipid Intercalators Covalent Protein Tags Genetic Markers Radio-labels Desired Characteristic PKH Dyes Dil, DiO CFSE Biotin-SE GFP, b-Gal 111In, 51Cr Fast, simple labeling +++ varies +++ ++ – + Intense labeling ++++ varies ++++ ++++ varies + Uniform and reproducible +++ varies +++ +++ + + Applicable to most cell types ++++ varies +++ +++ varies varies Not immunogenic ++++ ++++ + + ++ ++++ No effect on cell receptors or ++++ ++ varies varies + – functions Stable over many generations ++ ++ ++ + +++ – Immediate proliferation ++++ ++++ 24-48 hr delay 24-48 hr delay – varies monitoring possible Easy to monitor distribution + + + + +/– +++ in vivo Table 1. Characteristics of Cell Tracking Reagents. Abbreviations: Dil = 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate; DiO = 3,3’-dioctadecyloxacarbocyanine perchlorate; CFSE = carboxyfluorescein succinimidyl ester; Biotin-SE = biotin-succinimidyl ester; GFP = green fluorescent protein; b-Gal = b-galactosidase sigma-aldrich.com/biofiles 23

Intense, Reproducible Membrane Staining PKH dye labeling protocols can usually be optimized to give over 75% viable labeled cells with mean fluorescence more than 1,000 times background (see Figure 1). Such high signal-to-noise ratios are especially important when tagging proliferating cell lines because each daughter cell gets only half its parent’s label. With PKH dyes, up to ten generations can be followed before the intensity of the label falls near background (see Figure 2). Figure 3. Confocal assay for phagocytosis of malignant lymphocytes by monocytes-derived macrophages. Macrophages (labeled green with FITC), malignant lymphocytes (stained red with PKH26), and bispecific antibodies were combined and incubated at 37 °C. Confocal sections were examined after 30, 60 and 360 minutes and illustrate the process of target binding,

ingestion, and destruction. Ely, P., et al., “Bispecific-armed interferon gamma PKH Linker Kits primed macrophage mediated phagocytosis of malignant non-Hodgkins lymphome,” Blood, 87, 3813 (1996). Reprinted with permission.

PKH26 is complementary to the green PKH dyes. It can be quan- titated without interference from PKH2, PKH67, or fluorescein- tagged antibodies in studies of mixed cell populations (see Figure 3).

Methods Figure 1. Deconvoluted histogram of PKH26-labeled human peripheral blood leukocytes (PBL) on Day Zero showing non-proliferated control parent For General Cell Labeling population. The in vitro general cell labeling procedure can be used for monocytes, macrophages, lymphocytes, and other cells in suspension. Cells grown on the surface of tissue culture vessels may be stained in situ but heterogeneous staining may result. For homogeneous staining, adherent cells should first be suspended with a proteo- lytic treatment (e.g., trypsin + EDTA). Figure 4 represents the protocol schematically. A 23 cell suspension and a 23 dye solution, both in the PKH diluent supplied with the kit, are mixed and incubated briefly at room temperature. The labeling reaction is stopped by addition of protein (medium with serum or BSA). Labeled cells are washed 3-5 times to remove unbound dye. General cell labeling should be performed prior to monoclonal antibody staining to avoid capping the antibody with Figure 2. Deconvoluted histogram of CD4+/CD45RO+ subset in PKH26- the dye. labeled PBL activated with 2.5 µg/mL PHA for 6 days. Inset shows gates set For Phagocytic Cell Labeling by light scatter and immunophenotype. Phagocytic cells can be selectively labeled in the prescence of Characteristics of PKH Dyes non-phagocytic cells by using an alternate diluent. In this diluent, dyes do not dissolve, but instead form microaggregates; these Three different PKH dyes are used for labeling cells – PKH2, microaggregates cannot become embedded in membrane, but can PKH26, and PKH67. PKH2 and PKH67 are green fluorescent labels; be ingested by phagocytosis. This method has been used to study both can be detected with microscopes and flow cytometers lifetime and migrational patterns of macrophages and neutrophils equipped with fluorescein filters. Since their emission spectra have in vivo. little overlap with the red region, they are ideal for cytotoxicity studies4,5 in conjunction with red fluorescent viability probes like For analysis of proliferation based on the dye dilution profile, we propidium iodide (PI) and 7-aminoactinomycin D (7-AAD), as well recommend the Proliferation Wizard module in the Modfit soft- as with other red labels like R-phycoerythrin and Texas Red. They ware (Verity Software House, Topsham, ME). are also excellent for plant studies because they avoid interference from chlorophyll autofluorescence in the red region. Summary The in vivo half-life of PKH2 is 5-8 days, making it excellent for PKH Fluorescent Cell Linker Kits use patented fluorescent cell short- to medium-term studies. PKH67 has a longer aliphatic tail linker technology to incorporate reporter molecules into the cell than PKH2, resulting in more stable labeling and less cell-to-cell membrane. Labeled cells retain both biological and proliferative sigma-aldrich.com/biofiles transfer.6 It is expected to have an in vivo half-life of 10-12 days. activity, and are ideal for cell tracking and cell-cell interaction stud- Previous users of PKH2 may find that PKH67 gives even better ies. Due to the nonspecific labeling of the dyes, a wide variety of results for longer-term studies and for studies in which it is essen- cell types have been labeled successfully. The linkers have been tial to minimize cell-to-cell transfer. applied to both animal and plant cells. Table 2 lists common cell types that have been successfully labeled and Table 3 lists many PKH26 is a red fluorescent dye. It offers the longest in vivo half- PKH dye applications. life – greater than 100 days – making it ideal for in vivo cell tracking, cell proliferation studies, and other long-term assays.7-9 Rhodamine or phycoerythrin (PE) filters are suitable for PKH26 detection. Standard fluorescein excitation wavelengths may be used, but fluorescence intensity will be somewhat reduced. 24 PKH Linker Kits

In vitro: In vivo: Apoptosis (in conjunction with Annexin V-FITC) Migration Differentiation Homing Drug sensitivity/resistance Engraftment Cell-cell communication Immune reconstitution Antigen-driven proliferation Proliferation Conjugate formation, adhesion, and/or fusion Growth control Cellular cytotoxicity Differentiation Bystander killing Embryogenesis Antitumor immunity Adhesion Effect of vaccination on frequency of antigen Blood flow specific precursors Antitumor immunity Phagocytosis Antigen presentation Table 3. Monitoring Cell Functions Using PKH dyes.

References: 1. Samlowski, W.E., et al., J. Immunol. Methods, 144, 101 (1991). 2. The patented PKH dye technology, developed by Zynaxis Cell Science, is now owned by Phanos Technologies. 3. Horan, P.K., et al., Methods in Cell Biology, 33, 469 (1990). 4. Slezak, S. and Horan, P., J. Immunol. Meth., 177, 205 (1989). 5. Hatam, L., et al., Cytometry, 16, 59 (1994). 6. Unpublished data, Zynaxis, Inc.

PKH Linker Kits 7. Horan, P. and Slezak, S., Nature, 340, 167 (1989). 8. Yamamura, Y., et al., Cell. Molec. Biol., 41, S121 (1995). 9. Wallace, P., et al., Cancer Res., 53, 2358 (1993).

Ordering Information Cat. No. Name Pack Size PKH26GL PKH26 Red Fluorescent Cell Linker Kit 1 kit for General Cell Labeling MINI26 PKH26 Red Fluorescent Cell Linker Mini 1 kit Kit for General Cell Labeling PKH26PCL PKH26 Red Fluorescent Cell Linker Kit 1 kit for Phagocytic Cell Labeling PKH67GL PKH67 Green Fluorescent Cell Linker Kit 1 kit for General Cell Labeling MINI67 PKH67 Green Fluorescent Cell Linker 1 kit Mini Kit for General Cell Labeling PKH2GL PKH2 Green Fluorescent Cell Linker Kit 1 kit for General Cell Labeling PKH2PCL PKH2 Green Fluorescent Cell Linker Kit 1 kit for Phagocytic Cell Labeling

Figure 4. Standard protocol for PKH dye labeling. For additional information and references on PKH Hematopoietic cells: Bacteria, viruses, and parasites: CD34+ stem cells, CFU-s, bone mar- Bacillus subtilis, Haemophilus somnus, Cell Linker Dyes, please visit our Web site at row transplant “facilitating cells”, long Leishmania donovani, Listeria monocy- sigma.com/pkh. term culture initiating cells, primitive togenes, Plasmodium gallinaceum, progenitor cells, stromal cells Other Salmonella typhimurium, blood cells: Erythrocytes, neutrophils, Schistosoma mansoni, HIV, platelets Trichinella spiralis, Vibrio cholerae Immune cells: Non-mammalian cells/organisms: Lymphocytes, monocytes, macro- Dictyostelium discoideum, Newt lens- phages, thymocytes, splenocytes, cord regenerating cells, Rana catesbeiana blood T cells, cytotoxic T cells, autoim- (bull frog) red blood cells, Zebrafish mune T cells, NK cells, Langerhans and embryos dendritic cells Other primary cells: Plant Cells: Chick germ cells, embryonic cells, Protoplasts, Phytoplankton fibroblasts, epithelial cells, endothelial cells, leukemic blasts, mast cells, mes- enchymal cells, myoblasts, neurons and neuronal precursors, smooth muscle cells Cultured cells and cell lines: Miscellaneous particles: Erythrocyte Hybridomas, T cell lines and clones, ghosts, Fluorocarbon emulsions, tumor cell lines (K562, HL60) Liposomes Table 2. Cell Types Successfully Labeled with PKH Dyes. sigma-aldrich.com/biofiles 25 Fluorescent Microparticles and Nanobeads

Microparticles have been found to have widespread applications Unmodified MF particles have a hydrophilic, charged surface due in medicine, biochemistry, colloid chemistry, and aerosol research. to the high density of polar triazine-amino and -imino groups. The Uses include chromatographic separation media, supports for surface functional groups (methylol groups, amino groups, etc.) immobilized enzymes, and spacers in liquid crystal displays. allow covalent attachment of other ligands. For special applica- Fluorescently labeled microparticles are useful as standards in flow tions, the MF particles can be modified by incorporation of other cytometry, confocal laser scanning microscopy, and with light scat- functionalities such as carboxyl groups. This increases possible sur- tering instruments. They also have been used in environmental face derivatization such as chromophore or fluorophore labeling. science as tracers in flow measurements of gases and liquids, such Melamine resin microspheres are available as white particles or as laser Doppler anemometry (LDA), particle dynamic analysis with internally incorporated fluorescent labels. Both particle types (PDA), and particle image velocimetry (PIV). are available with either unmodified or carboxylated surfaces.

Fluorescently Labeled and Carboxylate-Modiﬁ ed

Melamine Microparticles Nanobeads Fluorescent Microparticles and Fluorescent melamine resin microspheres can be prepared with variations in size, type of fluorochrome, and surface functional groups. Typical dyes used in MF fluorescent particles are:

• FITC, green fluorescence λ λ ( Ex = 506 nm, Em = 529 nm) • Rhodamine B, orange fluorescence λ λ ( Ex = 560 nm, Em = 584 nm) • Nile Blue A, red fluorescence λ λ Figure 1. Fluorescence microscopic image of 10 mm melamine resin par- ( Ex = 636 nm, Em = 686 nm). ticles labeled with 7-amino-4-methylcoumarin (reproduced with permission of Microparticles GmbH). Outstanding characteristics of fluorescent MF particles (see Melamine Resin Particles Figure 2) are their narrow size distribution and an intense color and fluorescence. The homogeneously volume-stained particles Sigma offers a new generation of monodisperse polymer micro- have no leaching of the internally incorporated fluorochromes and spheres (see Figure 1) based on melamine resin (MF). Melamine they show high stability in organic solvents, comparable to white resin microspheres are manufactured by acid-catalyzed hydrother- MF particles. Fluorescent MF beads are also available with carbox- mal polycondensation of methylol melamine in the temperature ylate-modified surfaces containing a high density of functional range of 70-100 °C without any surfactants. By adjusting the groups (>0.1 mmole per gram of resin). pH value, the concentration of methylol melamine, and the reaction temperature, monodisperse particles with a predictable size between 0.5-15 mm can be produced in a one-pot synthesis. With excellent physical and chemical properties, melamine resin particles offer many advantages over other conventional polymer particles.

Physical and Chemical Properties of Melamine Resin Particles • Density: 1.51 g/cm3 • Refractive Index: 1.68

• Excellent monodispersity (C.V. <3%) and highly uniform spheri- Figure 2. Fluorescence microscopy image of FITC-labeled MF particles cal shape (shown as green) and Rhodamine B-labeled MF particles (shown as red). • Hydrophilic surface • High crosslinking density • High thermostability up to 300 °C For a complete list of our polymer-based micropar-

• Superior mechanical strength ticles and nanobeads, please visit our Web site at sigma-aldrich.com/biofiles • Stable and insoluble in acids and bases sigma.com/fluorescence. • Extremely high stability in organic solvents, no swelling or shrink- ing upon contact with organic solvents • Outstanding long-term stability in dispersions; no additives or stabilizers required • Aqueous suspensions are stable to repeated freeze-thaw cycles • Particles can be dried directly from their aqueous dispersions • Free flowing powders of dried particles can be redispersed in any dispersing agent without agglomeration 26 Fluorescent Microparticles and Nanobeads

Fluorescent Microparticles Ordering Information Cat No. Name Size (µm) Modification Fluorescence Dye Pack Size 86296 Microparticles based on melamine resin 1 Carboxylated FITC-marked 5 mL, 10 mL 88486 Microparticles based on melamine resin 3 Carboxylated FITC-marked 5 mL, 10 mL 90287 Microparticles based on melamine resin 10 Carboxylated FITC-marked 5 mL, 10 mL 73275 Microparticles based on melamine resin 1 Carboxylated Nile blue-marked 5 mL, 10 mL 74448 Microparticles based on melamine resin 3 Carboxylated Nile blue-marked 5 mL, 10 mL 73346 Microparticles based on melamine resin 6 Carboxylated Nile blue-marked 5 mL, 10 mL 76083 Microparticles based on melamine resin 10 Carboxylated Nile blue-marked 5 mL, 10 mL 74279 Microparticles based on melamine resin 1 Carboxylated Rhodamine B-marked 5 mL, 10 mL 73114 Microparticles based on melamine resin 3 Carboxylated Rhodamine B-marked 5 mL, 10 mL 83088 Microparticles based on melamine resin 6 Carboxylated Rhodamine B-marked 5 mL, 10 mL 88893 Microparticles based on melamine resin 10 Carboxylated Rhodamine B-marked 5 mL, 10 mL 90305 Microparticles based on melamine resin 1 None FITC-marked 5 mL, 10 mL

and Nanobeads 90312 Microparticles based on melamine resin 2 None FITC-marked 5 mL, 10 mL 72439 Microparticles based on melamine resin 3 None FITC-marked 5 mL, 10 mL 72833 Microparticles based on melamine resin 4 None FITC-marked 5 mL, 10 mL 75908 Microparticles based on melamine resin 5 None FITC-marked 5 mL, 10 mL

Fluorescent Microparticles Microparticles Fluorescent 70917 Microparticles based on melamine resin 6 None FITC-marked 5 mL, 10 mL 82230 Microparticles based on melamine resin 7 None FITC-marked 5 mL, 10 mL 77041 Microparticles based on melamine resin 8 None FITC-marked 5 mL, 10 mL 94050 Microparticles based on melamine resin 10 None FITC-marked 5 mL, 10 mL 93887 Microparticles based on melamine resin 1 None Nile blue-marked 5 mL, 10 mL 92992 Microparticles based on melamine resin 3 None Nile blue-marked 5 mL, 10 mL 94272 Microparticles based on melamine resin 4 None Nile blue-marked 5 mL, 10 mL

Fluorescent Nanobeads (Nanoparticles) Base Materials The understanding of nanoscale systems has advanced rapidly Fluorescent nanobeads (nanoparticles) can be prepared from a over the last few years, and these exciting scientific developments variety of polymers, each providing certain advantages. are being translated into a new generation of high technology Polyacrylnitrile (PAN) nanoparticles are ideally suited for FRET appli- products and processes. Nanotechnology has the potential to cations. When labeled, they are highly fluorescent and extremely impact a wide range of applications, from chemicals to electronics, small (less than 30 nm in diameter). PAN has a low concentration to sensors, to advanced materials. Nanoparticles have even been of interfering substances and the nanoparticles are available with used as drug carriers for DNA. carboxylated or streptavidin modified surfaces. All particles are One major aspect of nanotechnology is the development of fluo- supplied as a 0.5% (w/w) buffered aqueous suspension (10 mM rescent nanoparticles, which may have applications in optical data MES, pH 7). storage and the areas of biochemistry, bioanalytics, and medicine. PD is a novel polymer offering properties similar to polystyrene, Current fluorescent imaging methods are primarily based on dye but with much lower oxygen permeability, which results in a markers, which have limited photostability and light emission per higher photostability for most dyes. Particle size is ~40 nm and molecule. Nanoparticles overcome these limitations by offering the particle surface can be carboxylated or otherwise modified. All more intense and stable fluorescent signals. Fluorescently labeled particles are supplied in a 0.5% (w/w) buffered aqueous suspen- nanoparticles have been successfully used in a variety of immuno- sion (10 mM MES, pH 7). assays. Some of the limiting factors in latex agglutination assays can be avoided by using very small particles, which reduce the Custom Particles background absorbance and provide better colloidal stability. Custom microparticles and nanobeads with specific modifica- tions such as streptavidin are also available through Sigma. Please inquire at your local Sigma office for custom microparticles and nanobeads. sigma-aldrich.com/biofiles 27

Fluorescent Nanobeads Ordering Information Cat No. Name Size (nm) Modification Fluorescence Dye Pack Size 43187 Nanobeads based on PD 40 None Chromeon 470-marked 5 mL, 10 mL 69298 Nanobeads based on PD 40 Carboxylated Chromeon 470-marked 5 mL, 10 mL 66392 Nanobeads based on PD 40 None Chromeon 545-marked 5 mL, 10 mL 96151 Nanobeads based on PD 40 Carboxylated Chromeon 545-marked 5 mL, 10 mL 74036 Nanobeads based on PD 40 Carboxylated streptavidin Chromeon 545-marked 1 mL 83394 Nanobeads based on PD 40 None Chromeon 642-marked 5 mL, 10 mL 56559 Nanobeads based on PD 40 Carboxylated Chromeon 642-marked 5 mL, 10 mL 91915 Nanobeads based on PD 40 Carboxylated streptavidin Chromeon 642-marked 1 mL Nanobeads Fluorescent Microparticles and 43233 Nanobeads based on PAN 30 Carboxylated Chromeon 470-marked 5 mL, 10 mL 43666 Nanobeads based on PAN 30 None Chromeon 470-marked 5 mL, 10 mL 91402 Nanobeads based on PAN 30 Biotin-modified Chromeon 470-marked 1 mL 65779 Nanobeads based on PAN 30 None Chromeon 545-marked 5 mL, 10 mL 28499 Nanobeads based on PAN 30 Carboxylated Chromeon 545-marked 5 mL, 10 mL 80347 Nanobeads based on PAN 40 Streptavidin-modified Chromeon 545-marked 1 mL 66019 Nanobeads based on PAN 30 Carboxylated Chromeon 642-marked 5 mL, 10 mL 06245 Nanobeads based on PAN 30 Streptavidin-modified Chromeon 642-marked 1 mL 69030 Nano particles two-colored, 40 None Two-colored 1 mL calibration standard

References: 1. Heuberger, R., et al., Biofunctional Polyelectrolyte Multilayers and 17. Sukhorukov, G.B., et al., Stepwise Polyelectrolyte Assembly on Particle Microcapsules: Control of Non-specific and Bio-specific Protein Adsorption, Surfaces: A Novel Approach to Colloid Design, Polym. Adv. Technol., 9, 1-9 Adv. Funct. Mater., 15, 357-366 (2005). (1998). 2. Sukhorukov, G.B., et al., Comparative Analysis of Hollow and Filled 18. Stoffels, E., et al., Treatment of Dust Particles in an RF Plasma Polyelectrolyte Microcapsules Templated on Melamine Formaldehyde and Monitored by Mie Scattering Rotating Compensator Ellipsometry Swinkels, Carbonate Cores, Macromol. Chem. Phys., 205, 530-535 (2004). G.H.P.M., Pure & Appl. Chem., 70, 1151-1156 (1998). 3. Duchense, T.A., et al., Encapsulation and Stability Properties of 19. Caruso, F., et al., Nanoengineering of Inorganic and Hybrid Hollow Nanoengineered Polyelectrolyte Capsules for Use as Fluorescent Sensors, Spheres by Colloidal Templating, Science, 282, 1111-1114 (1998). Sensors Materials, 14, 293-308 (2002). 20. Caruso, F., et al., Production of Hollow Microspheres from 4. Park, M.-K., et al., Cross-linked Luminescent Spherical Colloidal and Nanostructured Composite Particles, Chem. Mater., 11, 3309-3314 (1999). Hollow-Shell Particles, Langmuir, 17, 7670-7674 (2001). 21. Caruso, F., et al., Core-Shell Particles and Hollow Shells Containing 5. Yang, W., et al., Layer by Layer Construction of Novel Biofunctional Metallo-Supramolecular Components, Chem. Mater., 11, 3394-3399 (1999). Fluorescent Microparticles for Immunoassay Application, J. Coll. Interface 22. Susha, A.S., et al., Formation of Luminescent Spherical Core-Shell Sci., 234, 356-362 (2001). Particles by the Consecutive Adsorption of Polyelectrolyte and CdTe(S) 6. Meier, W., Polymer Nanocapsules, Chem. Soc. Rev., 29, 295-303 (2000). Nanocrystals on Latex Colloids, Colloids Surf. A, 163, 39-44 (2000). 7. Rogach, A.L., et al., Nano- and Microengineering: 3-D Colloidal Photonic 23. Caruso, F., Hollow Capsule Processing through Colloidal Templating and Crystals Prepared From Sub-µm-sized Polystyrene Latex Spheres Pre-Coated Self-Assembly, Chem. Eur. J., 6, 413-419 (2000). with Luminescent Polyelectrolyte/Nanocrystal Shell, Adv. Mater., 12, 333- 24. Abbott, A., Space-Station Airlock to Serve as Temporary Lab, Nature, 337 (2000). 405, 7 (2000). 8. Moehwald, H., From Langmuir Monolayers to Nanocapsules, Colloids 25. Caruso, F., et al., Assembly of b-Glucosidase Multilayers on Spherical Surf., A, 172, 25-31 (2000). Colloidal Particles and their Use as Active Catalysts, Colloids Surf. A, 196, 9. Lerche, K.-H., and Kretzschmar, G., Effectiveness of Purification 287-293 (2000). Techniques in Cleaning Polymer Dispersions, Material Sci. Forum, 356, 25- 26. Moya, S., et al., Lipid Coating on Polyelectrolyte Surface Modified 26 (1988). Colloidal Particles and Polyelectrolyte Capsules, Macromolecules, 33, 4538- 10. Pelssers, E. and Lerche, K.-H., A New Red Fluorescent M/F-Latex Particle 4544 (2000). for Single Particle Optical Sizing, Colloids and Surfaces, 34, 241 (1989). 27. Voigt, A., et al., Membrane Filtration for Microencapsulation and 11. Schnablegger, H. and Glatter, O.J., Simultaneous Determination of Size Microcapsules Fabrication by Layer-by-Layer Polyelectrolyte Adsorption, Ind. Distribution and Refractive Index of Colloidal Particles from Static Light- Eng. Chem. Res., 38, 4037-4043 (2000). Scattering Experiments, Colloid Interface Sci., 158, 228-242 (1993). 28. Sukhorukov, G.B., et al., Microencapsulation by Means of Step-Wise 12. Thomas, H., et al., Plasma Crystal: Coulomb Crystallization in a Dusty Adsorption of Polyelectrolyte, J. Microencapsulation, 17, 177-185 (2000).

Plasma, Phys. Rev. Lett., 73, 652 (1994). 29. Sukhorukov, G.B., et al., Controlled Precipitation of Dyes into Hollow sigma-aldrich.com/biofiles 13. Porwol, T., et al., Three-Dimensional Imaging of Rhodamine 123 Polyelectrolyte Capsules Based on Colloids and Biocolloids, Adv. Mater., 12, Fluorescence Distribution in Human Melanoma Cells by Means of Confocal 112-115 (2000). Laser Scanning Microscopy, Acta Anat., 157, 116-125 (1996). 30. Leporatti, S., et al., Scanning Force Microscopy Investigation of 14. Guebel, G., et al., Micron and Sub-micron Aerosol Sizing with a Polyelectrolyte Nano- and Microcapsule Wall Texture, Langmuir, 16, 4059- Standard Phase-Doppler Anemometer, Aerosol Sci., 29, 1063 (1998). 4063 (2000). 15. Caruso, F., et al., Influence of Polyelectrolyte Multilayer Coating on 31. Radtchenko, I.L., et al., Assembly of Alternated Multivalent Ion/ Foerster Resonance Energy Transfer between 6-Carboxy-fluorescein and Polyelectrolyte Layers on Colloidal Particles. Stability of the Multilayers and Rhodamine B-labeled Particles in Aqueous Solution, J. Phys. Chem. B, 162, Encapsulation of Macromolecules into Polyelectrolyte Capsules, J. Colloid 2011-2016 (1998). Interface Sci., 230, 272-280 (2000). 16. Sukhorukov, G.B., et al., Layer-by-Layer Self Assembly of Polyelectrolytes 32. Caruso, F., Nanoengineering of Particle Surfaces, Adv. Mater., 13, 11-22 on Colloidal Particles, Colloids Surf. A 137, 253-266 (1998). (2001). 28 Books

Reference Books for Glycobiology

Sigma’s SciBookSelect™ offers the Best Books for the Best Minds. Our selected library of 2,000+ titles helps you keep pace with new technology while updating your protocols and knowledge. Whether your area of interest includes Molecular Biology, Drug Discovery, Proteomics, Analytical Chemistry, Cell Culture, Organic Chemistry, Spectroscopy, Cell Signaling, Materials Science, Medicinal Chemistry, Chromatography or Spectral Libraries, SciBookSelect can help you find the right book or CD. Visit us at sigma-aldrich.com/books. Books

Z705969 Principles of Fluorescence Spectroscopy 3rd ed. Z705683 Fluorescent Energy Transfer Nucleic Acid Probes: The third edition of the established classic text reference, will Designs and Protocols enhance upon the earlier editions’ successes. It will maintain the In the first comprehensive treatment of energy transfer (ET) emphasis on basics, while updating the examples to include recent nucleic acid probes, experts thoroughly describe all the major results from the literature. This edition also includes new chapters probes, both fluorescence resonance energy transfer (FRET)-based on single molecule detection, fluorescence correlation spectros- and non-FRET-based, and provide a complete set of techniques to copy, novel probes and radiative decay engineering. monitor DNA and RNA reactions, including hybridization, ampli- J. Lakowicz, Springer, 2006, 958 pp., hard cover fication, cleavage, folding, and associations with proteins, other molecules, and metal ions. Optimal design strategies for customized ET probes are presented, as well as techniques for distance determination in protein–DNA complexes and the detection of topological DNA alterations, mutations, DNA breaks and single nucleotide polymorphisms. Authors describe in detail the design and application of ET-using molecular devices, such as biosensors, molecular machines, and logic gates for molecular scale computa- tion. V. Didenko, Humana Press, 2006, 392 pp., hard cover

Detection Web Resource Over 1000 Detection Find the Optimal Probe Products or Label! - Stains, Dyes, & Markers - Sortable tables — search by - Fluorescent Micro- & excitation or emission wavelength Nanoparticles - Cross reference guide — fi nd - Standards & Kits products suitable for a specifi c - Chemiluminescent Compounds detection methodology - Scintillation Reagents - Reagents for Luminescence - Browse unique labels — with - Fluorescent Rotors enhanced sensitivity, large wave- - And more... length gaps, or long fl uorescence sigma.com/fl uorescence lifetimes sigma-aldrich.com/biofiles The shape of Sigma Life Science

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