Protein Quantification and Qualification Application Compendium Protein Quantification Using the Nanodrop One Spectrophotometer

Protein quantification and qualification Application compendium Protein quantification using the NanoDrop One Spectrophotometer

Welcome to protein quantification and qualification e-book. Here you’ll find a compendium of useful technical documents, application notes, and protocols for measuring proteins using the Thermo Scientific™ NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer.

Scientists can use the NanoDrop One/OneC instrument to quantify the protein content in their sample using either direct or indirect measurements. An example of a direct measurement is the Protein A280 application, which calculates protein concentration based on the sample absorbance at 280 nm and the protein- and wavelength-specific extinction coefficient. Colorimetric assays are an example of an indirect measurement. The NanoDrop One/OneC Software is hardcoded with applications to measure the product of the BCA, Bradford, Lowry, and Pierce™ 660 nm reactions.

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Table of contents

Blanking with high absorbing buffers such as RIPA negatively affects Protein A280 measurements ...... 3

NanoDrop One educational animations ...... 6

Quantify protein and peptide preparations at 205 nm ...... 7

On-demand webinar: Protein Sample Evaluation using the NanoDrop One UV-Vis Spectrophotometer ...... 10

BCA protein assay ...... 11

Pierce Rapid Gold Bca Protein Assay ...... 14

Bradford protein assay ...... 17

Lowry protein assay ...... 20

Pierce 660 nm protein assay ...... 23

A custom method for hemoglobin measurements ...... 26

 TECHNICAL NOTE No. T-145 No.

Blanking with high absorbing buffers such as RIPA negatively affects Protein A280 measurements

Abstract Thermo Scientific™ NanoDrop™ Microvolume UV-Vis Spectrophotometers are useful analytical tools for measuring the absorbance of small volumes of sample. Before making a sample measurement, the instrument must be blanked. The best practice for making a blank measurement is to use the buffer in which the sample is suspended. Doing so allows the software to account for the absorbance of the buffer solution and report an accurate analyte absorbance. The best buffers for UV-Vis spectroscopy have minimal absorbance at the analysis wavelength of the analyte. Here we show that blanking with a buffer that absorbs an appreciable amount of light leads to atypical absorbance measurements by Thermo Scientific NanoDrop One/OneC diminishing the amount of light available for the sample measurement. Microvolume UV-Vis Spectrophotometer

Introduction The most basic components of a UV-Vis spectrophotometer include a light source, the sample, and a detector. Light is illuminated through the sample and the amount of light transmitted to the detector is quantified. To calculate the absorbance of a sample, the amount of light transmitted through the sample must be compared to the amount of light transmitted through a reference substance called the blank. Given the sample and blank transmittance, one can calculate the absorbance of the sample with the equation:

The function of the blank measurement is to remove the absorbance contributed to the sample measurement by the buffer in which the sample is suspended. Essentially, the blank measurement allows the software to subtract the buffer signal out of the sample signal and accurately report the analyte absorbance. The above equation can be rewritten by expanding the logarithmic term to obtain:

 It is generally recommended to blank the instrument The Thermo Scientific™ NanoDrop™ One/OneC Microvolume with the buffer in which the sample is suspended; UV-Vis Spectrophotometer operating software is built however, not all buffers are suitable for absorbance with the Thermo Scientific™ Acclaro™ Sample Intelligence spectroscopy. A good buffer for blanking NanoDrop UV-Vis Technology. One of the new technologies is the blank Spectrophotometers should have no more than ± 0.04 AU absorbance verification feature. The software will not let you (absorbance units at a 10 mm pathlength) at the sample’s proceed if you try blanking with a buffer that has increased analytical wavelength. The analytical wavelength refers to absorbance at the analysis wavelength. Instead the software the wavelength whose absorbance is used to calculate the will display the message “Error: Blank solution absorbance analyte concentration. When the software automatically too high. Clean both pedestals and blank again.” This check selects the best pathlength for the measurement, it does is in place because blanking with a buffer such as RIPA can so using the absorbance at the analytical wavelength. negatively affect the absorbance measurement.

Radioimmunoprecipitation assay buffer (RIPA) is frequently Measuring protein suspended in PBS and RIPA used in protein lysis preparations, but absorbs a large The Protein A280 application measures the absorbance amount of light near 280 nm. In this application note, peak at 280 nm and calculates a protein concentration we examine the effects of blanking with RIPA on the using the protein-specific extinction coefficient. The measurement of protein absorbance at 280 nm. absorbance peak at 280 nm appears due to the presence of tryptophan, tyrosine, and cysteine double bonds in the The absorbance spectrum of RIPA buffer protein. Two solutions were prepared with the same weight When preparing to make absorbance measurements of BSA and the same volume of solvent – one with PBS of an analyte in a buffer one has never used before, it is and the other with RIPA. Theoretically, the software should recommended to perform a buffer analysis to determine calculate the BSA concentration in both samples to be how much light the buffer absorbs. A buffer analysis is very equivalent. Figure 2 displays the two resulting absorbance quick to perform. Simply open the application in which spectra. The black spectrum shows BSA suspended you will make analyte measurements, blank the instrument in PBS after the instrument was blanked with PBS. The with water, and measure your buffer as if it were a sample. red spectrum shows the result of blanking with RIPA As stated above, you want the buffer to have minimal and measuring BSA suspended in RIPA. While the PBS absorbance. spectrum appears as we would expect, the RIPA spectrum is quite distorted. The BSA sample suspended in PBS Figure 1 shows the absorbance spectrum of RIPA after displays a peak at 280 nm, yet the BSA sample suspended running a buffer analysis. RIPA absorbs approximately in RIPA displays a trough at 280 nm. The difference in the 22 AU at 280 nm, the analytical wavelength of proteins. By absorbance at 280 nm between these two samples is comparison, phosphate buffered saline (PBS) absorbs less approximately 88%. than 0.01 AU at 280 nm (not pictured).

 Figure 2: Absorbance spectra of BSA suspended in PBS and RIPA. The black spectrum shows a pure BSA sample suspended in PBS after the instrument was blanked with PBS. The red spectrum was generated by blanking the instrument with RIPA and measuring BSA in RIPA buffer.

 Figure 1: Buffer analysis of RIPA. RIPA absorbs approximately 22 AU at 280 nm, the analytical wavelength of proteins.

 RIPA buffer limits the light intensity available for a Conclusion sample measurement It is recommended to blank NanoDrop UV-Vis The intensity check diagnostic is designed to confirm Spectrophotometers using the same buffer in which the flash lamp and spectrometer are working within the sample is suspended. To make accurate analyte specifications. When running the intensity check, the measurements, the blank buffer should have minimal user leaves the pedestals dry and the detector measures absorbance near the analyte analytical wavelength. This the amount of light produced by the lamp at a 1 mm is true with the Protein A280 application as well as with pathlength. The software verifies the peaks in the xenon other applications, such as Nucleic Acids. Researchers can spectrum appear at their NIST-traceable locations. Three determine the buffer absorbance by performing a buffer intensity checks were performed – one with water on the analysis. In the case of RIPA buffer, it absorbs a large amount pedestals, one with PBS on the pedestals, and one with of light at the analytical wavelength of proteins. Absorbing RIPA on the pedestals. Figure 3 shows the resulting light such high amounts of light limits the light available for the intensities from 220 – 350 nm when different solvents were analyte measurement. This characteristic makes RIPA a poor present on the pedestals. buffer choice for quantifying protein concentration using a direct 280 nm measurement. Rather than using the Protein Deionized water is highly transparent across the UV-Vis A280 application, it is recommended to use a colorimetric spectrum. Conversely, RIPA absorbs a large amount of assay application in the NanoDrop Spectrophotometer light, especially in the low UV region. Data presented here Software to measure the concentration of proteins in show the water (black solid line) and PBS (red dashes) RIPA buffer. The operating software for full spectrum intensities overlap. This tells us the light signal is not NanoDrop UV-Vis Spectrophotometers are hardcoded with attenuated by PBS buffer from 200 – 350 nm. In contrast, applications to read the results of the Bradford, BCA, and the intensity of RIPA (black dashes) shows a complete Thermo Scientific™ Pierce™ 660 nm Protein Assays. This is attenuation of light with wavelengths 220 – 235 nm and discussed in detail in the technical document T112 – Influence 270 – 285 nm. of Buffer on Choice of Protein Quantification Method.

 Figure 3: Flash lamp intensities with different solvents on the pedestal. The intensity of the lamp with PBS on the pedestals (red dashes) is the same as the intensity with water on the pedestals (black solid line). The signal with RIPA on the pedestals (black dashes) is attenuated near 280 nm and at wavelengths 220 – 235 nm. For further assistance, contact NanoDrop technical support at [email protected] or visit thermofisher.com/nanodrop

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 APPLICATION NOTE Quantify protein and peptide No. AN52774 preparations at 205 nm NanoDrop One Spectrophotometer

Authors Sean Loughrey, Jessica Mannion, and Brian Matlock Thermo Fisher Scientific, Wilmington, DE

Abstract Life scientists can quantify peptide and protein samples on the Thermo Scientific™ NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometers using the A205 preprogrammed direct absorbance application. The new A205 application offers a choice of methods for peptides that contain Tryptophan and Tyrosine residues in their sequence as well as peptides that completely lack aromatic amino acids. The A205 application offers enhanced sensitivity for peptide quantification in seconds from only Figure 1: NanoDrop One Proteins Home screen showing available 2 µL of sample. preprogrammed applications for protein quantitation.

Introduction A protein’s peptide backbone absorbs light in the deep Researchers have always needed UV region (190 nm-220 nm), and this absorbance can be ways to quickly quantify various used for protein sample quantitation. The A205 protein biomolecules (e.g., protein and nucleic quantitation method has several advantages over the direct acid preparations) as a routine part A280 protein method such as lower protein-to-protein of their workflows. This information variability (because A205 extinction coefficients are not helps them make informed decisions based on amino acid composition) and higher sensitivity before proceeding with downstream (because of the high molar absorptivity proteins have at experiments. There are many protein 205 nm). However, technical limitations made it difficult to quantification methods to choose from obtain these measurements in the past. Spectrometers’ including gravimetric approaches, stray light performance, deep UV linearity, and protein colorimetric assays, direct spectrophotometric UV buffers containing UV-absorbing components have all measurements (such as A280), and amino acid analysis. added to the challenge of obtaining A205 data. The All of these methods have their strengths and weaknesses. NanoDrop One patented sample-rentention technology and Direct spectrophotometric microvolume UV measurements low stray light performance have simplified quantification of are a popular choice for researchers because they are small amounts of protein by A205 methods. simple to perform, require no reagents or standards, and consume very little sample. The Thermo Scientific In this application note, we discuss the three A205 NanoDrop One Spectrophotometer has preprogrammed measurement options included in the NanoDrop One applications (Figure 1) for direct quantification of proteins Protein A205 application and present performance data for using absorbance measurements at 280 nm and 205 nm. each option. This application note specifically describes how to use the Protein A205 application to quantify protein samples.  A205 extinction coefficients for peptide and protein A205 performance on the NanoDrop One measurements Preparations of polymyxin, a cationic detergent antibiotic The NanoDrop One Protein A205 application allows with a peptide backbone, but no Trp or Tyr residues, customers to choose from three different options (Figure 2). were made in 0.01% Brij® 35 buffer and measured on the The selected option will automatically determine the NanoDrop One and the Thermo Scientific™ Evolution™ 300 extinction coefficient that will be used to calculate the UV-Vis Spectrophotometers. To ensure the validity of the protein concentration based on the sample absorbance at measurements taken with the Evolution 300 instrument, 205 nm. the polymyxin preparations were diluted in 0.01% Brij buffer to ensure that the measurements taken were within • ε =31 method 205 the linear range of the detector. For measurements on the • Scopes method2 NanoDrop One instrument 2 µL of sample were pipetted directly on the sample pedestal, while a 10 mm quartz 1mg/mL • Other = custom method ε205 cuvette was used for measurements on the Evolution 300 Spectrophotometer. The polymyxin concentration Previous studies showed that most protein solutions at data obtained on both instruments (Table 1) were plotted 1mg/mL 1 mg/mL have extinction coefficientsε ( 205 ) ranging (Figure 3). Regression line shows that protein concentration 2 -1 -1 from 30 to 35 . The ε205 of 31 mL mg cm is an extinction results from the NanoDrop One instrument are in good coefficient often used for peptides lacking tryptophan agreement to the results obtained on a traditional high end and tyrosine residues1. The Scopes method gives a more UV-Vis spectrophotometer using a cuvette. accurate ε205, especially for proteins containing a significant amount of tryptophan (Trp) and tyrosine (Tyr) residues. NanoDrop One Evolution 300 The increased accuracy of this method takes into account Target [Conc] Std. [Conc] [Conc] A205 the significant absorbance at 205 nm contributed by the mg/mL Dev. mg/mL mg/mL aromatic side chains of Trp and Tyr. This method uses 0 -0.01 0.04 -0.18 -0.02 an A280/A205 ratio in its equation to correct for Trp and 5 0.11 0.01 3.60 5.05 3 Tyr side-chain absorbance . Recently, Anthis and Clore 10 0.27 0.01 8.84 10.53 proposed the use of a sequence-specificε 205 calculation 15 0.44 0.02 14.08 17.0 9 (e.g., custom/Other method), which is suitable for a wide 50 1.68 0.01 54.14 55.32 1 range of proteins and peptides . This method is appropriate 100 3.39 0.01 109.44 108.48 for pure preparations of proteins or peptides whose amino 200 6.64 0.03 214.16 222.50 acid sequences are known. Table 1: Various preparations of Polymyxin were measured on the NanoDrop One and Evolution 300 Spectrophotometers. Five(5) replicates of each solution were measured on the NanoDrop One instrument using the 205=31 application. Solutions with absorbance over 1.0A were diluted and measured in triplicate on the Evolution 300 instrument.

Figure 2: NanoDrop One Protein A205 methods selection screen.

Figure 3: Polymyxin concentrations calculated with the Evolution 300 and NanoDrop One instruments were plotted. Regression line shows that protein concentration measurements on the NanoDrop One instrument are in good agreement to those obtained on a traditional high end UV-Vis spectrophotometer.  # of Trp of Tyr

Protein [Concentration] [Concentration] Trp Tyr A205 STDV Preparation e205=31 (μg/mL) Scopes Method (μg/mL) BSA 1 3 21 3.960 0.013 127.73 131.80 BSA 2 3 21 37.271 0.218 1202.30 1261.71 BSA 3 3 21 70.044 0.239 2259.48 2387.91 BSA 4 3 21 129.170 1.458 4166.77 4345.20 BSA 5 3 21 271.027 0.851 8742.81 9198.13 Lysozyme 1 6 3 29.069 0.169 9 37.71 795.95 Lysozyme 2 6 3 53.651 0.545 1730.68 1459.05 Lysozyme 3 6 3 102.713 0.668 3313.32 2814.79 Polymyxin 1 0 0 0.112 0.015 3.60 3.12 Polymyxin 2 0 0 0.274 0.014 8.84 10.12 Polymyxin 3 0 0 0.437 0.021 14.08 16.03 Polymyxin 4 0 0 1.678 0.014 54.14 60.99 Polymyxin 5 0 0 3.393 0.014 109.44 125.16 Polymyxin 6 0 0 6.639 0.034 214.16 244.87

Table 2: Comparison of different A205 methods for various protein and peptide preparations on the NanoDrop One Spectrophotometer.

To assess the effect that the extinction coefficients used at and tyrosine residues has a large effect on the calculated

205 nm (i.e., Scopes and ε205=31 methods) would have on concentration (Table 2). This is because tryptophan is the the result, we prepared dilutions of three different proteins largest contributor to A280 absorbance, and the Scopes with varied amounts of aromatic residues: bovine serum method uses the A280/A205 ratio to correct for aromatic albumin (BSA, 3 Trp and 21 Tyr residues), lysozyme (6 side-chain absorbance at A205. Trp and 3 Tyr residues) and polymyxin (no Trp, no Tyr).

These preparations were measured on the NanoDrop One Our results show that A205 quantification using theε 205=31 instrument using the ε205=31 and Scopes methods (Table 2). method gives comparable results when proteins have only a few tryptophan residues. Conclusion To assess NanoDrop One Spectrophotometer performance One limitation of the A205 method is that many of protein at A205, we compared polymyxin concentration results buffers commonly used have absorbance at 205 nm. Before obtained with the NanoDrop One and the Evolution using this technique, we recommend checking the protein 300 benchtop Spectrophotometers, which have buffer for any contribution to the absorbance at 205 nm. excellent stray light performance. Table 1 shows that References the NanoDrop One instrument provided very consistent 1. Anthis, NJ and Clore, GM 2013. Sequence-specific determination of protein and peptide results between replicate measurements at 205 nm concentrations by absorbance at 205 nm. Protein Science 22:851-858. with standard deviations below 0.04A. In addition, the 2. Goldfarb, AR, Saidel, LJ, Mosovich E 1951. The ultraviolet absorption spectra of proteins. Journal of Biological Chemistry 193(1):397-404. results obtained with both instruments were comparable 3. Scopes, RK 1974. Measurement of protein by spectrophotometry a 205 nm. Analytical (Figure 3). Comparison between the A205 methods Biochemistry 59:277-282.

(Scopes and ε205=31 methods) offered in the NanoDrop One A205 application shows that the number of tryptophan

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For Research Use Only. Not for use in diagnostic procedures. ©2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. AN52774_E 05/19M  On-demand webinar Protein Sample Evaluation using the NanoDrop One UV-Vis Spectrophotometer

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 PROTOCOL NanoDrop One/OneC Spectrophotometers

BCA protein assay

Introduction Supplies The BCA Protein Assay combines the protein-induced Equipment: biuret reaction with the highly sensitive and selective • NanoDrop One/OneC Spectrophotometer colorimetric detection of the resulting cuprous cation (Cu1⁺) by bicinchoninic acid (BCA). A purple colored reaction • 0.5–2 µL pipettor (and low retention tips) and product is formed by the chelation of two molecules of 10–1000 µL pipettors BCA with one cuprous ion. The BCA/copper complex is water-soluble and the increase in absorbance is linear Materials: across a wide protein concentration range. In conjunction • Low lint laboratory wipes with the micro-volume capability of a Thermo Scientific™ NanoDrop™ Spectrophotometer, the assay provides an • 0.5 mL microcentrifuge tubes or 0.2 mL mini-centrifuge accurate means of protein quantitation with minimal strip tubes and caps consumption of sample. Reagents: Note: All specifications and protocol instructions • Thermo Scientific™ Pierce™ BCA Protein Assay Kit, presented below are specific for the pedestal mode of the product number 23225, 23227, or 23250 Thermo Scientific™ NanoDrop™ One/OneC instruments. ™ ™ Please refer to the reagent manufacturer for additional • Thermo Scientific Pierce BSA Standard Pre-Diluted guidance when utilizing the cuvette mode of the Set, product number 23208, or other commercially NanoDrop OneC Spectrophotometer. available protein standard • Thermo Scientific™ NanoDrop™ PR-1 Reconditioning Kit, Dynamic range PN CHEM-PR1-KIT The micro-assay has a linear range of 20–200 µg/mL using a 1:1 sample to reagent ratio. A higher range of Assay recommendations 125–2000 µg/mLmay be obtained using a 1:20 sample to • Measure 2 µL sample aliquots reagent ratio. • All standards and samples should be measured within 10 minutes

• Re-condition pedestals with PR-1 upon assay completion Thermo Scientific NanoDrop Onec Microvolume UV-Vis Spectrophotometer

 Sample preparation measurement of the zero reference and at least one 1. Equilibrate all reagents, unknowns and protein standard. It is recommended that additional standards standards to room temperature. Mix thoroughly but be included as necessary to cover the expected assay gently to avoid introducing micro bubbles. concentration range.

2. Prepare enough fresh working reagent for all standards 3. On the left side of the screen, select the Curve Type and samples to be measured using a 50:1 ratio of the and number of replicates to measure. We recommend kit reagents A:B. selecting the Linear curve type and 3 replicates. Tap Done. 3. Add the appropriate reagent volume to each microcentrifuge tube or PCR strip well. Note: If the instrument self-test begins, do not touch the instrument. Micro-assay (1:1 sample to working reagent ratio): Add 10 µL of working reagent to each standard 4. Establish a blank using diH2O. It is advisable to use and sample tube/well. the dye reagent and protein buffer without any protein added as the zero reference sample for this assay. High range assay (1:20 sample to working reagent ratio): Add 200 µL of working reagent to Pedestal Option: Pipette 2 µL of blank solution onto each standard and sample tube/well. the bottom pedestal, lower the arm and tap Blank.

4. Add 10 µL of standards or samples to the appropriate Cuvette Option (Model OneC only): Insert the cuvette tube. Mix well by gentle vortexing. If necessary, collect the noting the direction of the light path indicated by the solution at the bottom of the tube by a brief centrifugation. etched arrow. The optical beam (2 mm) is directed 5. Incubate the standard and sample tubes at either 8.5 mm above the bottom of the cuvette. Refer to the 37º C for 30 minutes or 60º C for ~ 5 minutes. Cool the cuvette manufacturer for volume recommendations. tubes to room temperature. Note: The arm must be down for all measurements, except those made with cuvettes. It is recommended that cuvettes be removed from the instrument prior to making a pedestal measurement to ensure that the pedestal arm can move to the proper starting position.

5. Follow the direction at the top of the screen to measure the reference and standards. After each measurement, wipe the upper and lower pedestals using a dry laboratory wipe. After your last standard measurement, you can choose to load more standards or run samples.

6. After all of the Standards have been measured, click on

Typical absorbance spectrum for a BCA protein assay sample the Run samples radio button. Enter a sample ID and measurement. load a 2 μL aliquot of sample when using the pedestal. Tap Measure. Protocol 1. Select the Protein tab from the Home screen. Tap the 7. After completing all Standard and Samples Protein BCA application button. measurements it is good practice to re-condition the pedestals using PR-1. 2. Enter the values for each standard concentration in the table on the right. The software allows for the reference 8. It is not necessary to blank the instrument between the and up to 7 additional standards. The zero reference and/ standard and the unknown sample measurements. or standards can be measured with up to 3 replicates. Note: A fresh aliquot of sample should be used for Note: The minimum requirement for standard curve each measurement. generation is the measurement of two standards or the

 Standard curve data

BSA A562 Std. % (µg/mL) (n=3) dev. CV 0 -0.077 0.016 NA 125 0.120 0.013 NA 250 0.327 0.010 NA 500 0.691 0.002 0.3 750 1.148 0.005 0.4 1000 1.524 0.008 0.5 1500 2.130 0.025 1.2 2000 2.759 0.013 0.5

Typical absorbance values for a High Range assay using 1:20 sample to reagent ratio assay using the Pierce BCA reagent.

For additional information regarding the BCA Protein assay and reagents: thermoscientific.com/pierce

Toll free (US & Canada): 877-724-7690 • Technical support: [email protected]

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Pierce Rapid Gold BCA Protein Assay

Introduction Supplies The Thermo Scientific™ Pierce™ Rapid Gold BCA Protein Equipment: Assay combines the protein-induced biuret reaction with • NanoDrop One/OneC Spectrophotometer the highly sensitive and selective colorimetric detection of the resulting cuprous cation (Cu1+) by a new, unique BCA • 0.5–2 µL pipettor (and low retention tips), 10–100 µL chelator. An adaptation of the traditional BCA assay, the pipettor, and a 100–1000 µL pipettor Rapid Gold BCA Protein Assay Kit has been optimized to develop in 5 minutes at room temperature. The Rapid Materials: Gold BCA Protein Assay produces an orange-gold colored • Low lint laboratory wipes reaction product, which is formed by the chelation of 2 molecules of the chelator with 1 cuprous ion. • 0.5 mL microcentrifuge tubes or 0.2 mL mini-centrifuge strip tubes and caps or 96 well PCR plate (for standards This water-soluble complex exhibits a strong absorbance and sample reactions) at 480 nm that is very linear with increasing protein concentrations over a broad working range of Reagents: 20–2000 µg/mL. In conjunction with the microvolume • Pierce Rapid Gold BCA Protein Assay Kit, PN A53225 or capability of a Thermo Scientific™ NanoDrop™ One/OneC A53227 Spectrophotometer, the assay provides an accurate means ™ ™ of protein quantitation with minimal consumption of sample. • Thermo Scientific Pierce BSA Standards Set (pre- diluted PN 23208 or standard ampules PN 23209), ™ ™ Note: All specifications and protocol instructions presented Thermo Scientific Pierce BGG Standards (pre-diluted below are specific the pedestal mode for the NanoDrop PN 23213 or standard ampules PN 23212), or other One instrument. Please refer to the reagent manufacturer commercially prepared protein standard for additional guidance when utilizing the cuvette mode of • Thermo Scientific™ NanoDrop™ PR-1 Reconditioning Kit, C the NanoDrop One Spectrophotometer. PN CHEM-PR1-KIT

Dynamic range Assay recommendations The Rapid Gold BCA Protein Assay has a linear range of • Measure 2 µL sample aliquots 20–2000 µg/mL using a 1:20 sample to reagent ratio. • Re-condition pedestals with PR-1 upon assay completion

Thermo Scientific NanoDrop Onec Microvolume UV-Vis Spectrophotometer

 Sample preparation 2. Select USB Drive and the select Load Method. Then 1. Equilibrate all reagents, unknowns and protein select the Pierce Rapid Gold BCA method file and tap standards to room temperature. Mix thoroughly but Load button. Then select Run Method. gently to avoid introducing micro bubbles. 3. Enter the values for each standard concentration in the 2. Prepare enough fresh working reagent (WR) for all table on the right. The software allows for the reference standards and samples to be measured using a and up to 7 additional standards. Tap Done. 50:1 ratio of the kit reagents A:B. Note: The minimum requirement for standard curve Note: When Rapid Gold BCA Reagent B is first added generation is the measurement of two standards or the to Rapid Gold BCA Reagent A, a pale blue precipitate measurement of the zero reference and at least one may be observed, but, upon vortexing or mixing for standard. It is recommended that additional standards < 5 seconds, the precipitate should dissolve to yield a be included as necessary to cover the expected assay clear, green solution. Use fresh working reagent each concentration range. Tap Done. time. Note: If the instruments self-test begins, do not touch 3. Add the appropriate WR reagent volume to each the instrument. microcentrifuge tube or PCR strip well. 4. Prepare a blank solution using 10µL buffer+100 µL WR. Rapid Gold BCA (1:10 sample to working reagent ratio): Add 100 µL of working reagent to each Pedestal Option: Pipette 2 µL of blank solution onto the standard and sample tube/well. bottom pedestal, lower the arm and tap Blank.

4. Add 10 µL of standards or samples to the appropriate Cuvette Option (Model OneC only): Insert the cuvette tube. Mix well by gentle vortexing. noting the direction of the light path indicated by the 5. Incubate the standard and sample tubes at room etched arrow. The optical beam (2 mm) is directed temperature for 5 minutes. 8.5 mm above the bottom of the cuvette. Refer to the cuvette manufacturer for volume recommendations. Note: It is important to measure all standards and samples within 10 minutes or stop the reactions by Note: The arm must be down for all measurements, adding 25 µL 1N HCl. except those made with cuvettes. It is recommended that cuvettes be removed from the instrument prior to making a pedestal measurement to ensure that the pedestal arm can move to the proper starting position.

5. Follow the direction at the top of the screen to measure the standards. After each measurement, wipe the upper and lower pedestals using a dry laboratory wipe.

6. After all of the Standards have been measured, enter a sample ID and load a 2 μL aliquot of sample when using the pedestal. Tap Measure.

7. After completing all Standard and Samples Typical absorbance spectrum for a Rapid Gold BCA protein assay measurements it is good practice to re-condition the sample measurement. pedestals using PR-1.

Protocol 8. It is not necessary to blank the instrument between the 1. Select the Custom tab from the Home screen. Download standard and the unknown sample measurements. Pierce Rapid Gold BCA method file from our website and save it on a USB drive. Insert USB drive into the Note: A fresh aliquot of sample should be used for instrument and tap the Custom application button. each measurement.

 Standard curve data

BSA (µg/mL) A480 125 0.446 250 0.851 500 1.495 1000 2.687 1500 4.021 2000 5.122

Typical absorbance values for an assay using 1:20 sample to reagent ratio assay using the Pierce Rapid Gold BCA reagent.

For additional information regarding the Rapid Gold BCA Protein assay and reagents: thermoscientific.com/pierce

Toll free (US & Canada): 877-724-7690 • Technical support: [email protected]

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Bradford protein assay

Introduction Dynamic range Use of the coomassie G-250 dye in a colorimetric reagent The Micro assay has a linear range of 15 - 100 µg/mL using for the detection and quantitation of total protein was first a 1:1 sample to reagent ratio. The Standard assay has a described by Dr. Marion Bradford in 1976. Protein binds higher range of 100 - 1000 µg/mL which may be obtained to the coomassie dye in the acidic environment of the using a 1:30 sample to reagent ratio. reagent. This results in a spectral shift from the reddish/ brown form of the dye (absorbance maximum at 465 nm) Supplies, materials & reagents to the blue form of the dye (absorbance maximum at • NanoDrop One/OneC Spectrophotometer 610 nm). The difference between the two forms of the dye is greatest at 595 nm, the optimal wavelength to measure • 0.5 - 2 µL pipettor (and low retention tips) and the blue color from the coomassie dye-protein complex. 10–1000 µL pipettors. In conjunction with the micro-volume capability of the • Low lint laboratory wipes Thermo Scientific™ NanoDrop™ Spectrophotometers, the assay provides an accurate means of protein quantitation • 0.5mL microcentrifuge tubes or 0.2 mL mini-centrifuge with minimal consumption of sample. strip tubes and caps

• Thermo Scientific™ Pierce™ Coomassie Plus Assay Kit, Note: All specifications and protocol instructions presented PN 23236 or 23238 below are specific to the pedestal mode of NanoDrop C One/One instruments. Please refer to the reagent • Thermo Scientific™ Pierce™ BSA Standard Pre-diluted manufacturer for additional guidance when utilizing the Set, PN 23208 or other commercially prepared protein C cuvette mode of the NanoDrop One Spectrophotometer. standard

• Thermo Scientific™ NanoDrop™ PR-1 Reconditioning Kit, PN CHEM-PR1-KIT

Assay recommendations • Measure 2 µL sample aliquots

• Making standard and sample measurements in triplicate is good practice, particularity with the limited assay signal obtained with the Bradford Assay.

• Re-condition pedestals with PR-1 upon assay completion

Thermo Scientific NanoDrop Onec Microvolume UV-Vis Spectrophotometer

 Sample preparation 4. Establish a blank using dH2O. 1. Equilibrate all reagents, unknowns and protein standards to room temperature. Mix thoroughly but Pedestal Option: Pipette 2 µL of blank solution onto gently to avoid introducing micro bubbles. the bottom pedestal, lower the arm and select Blank.

2. Add the appropriate reagent volume to each Cuvette Option (Model NanoDrop OneC only): microcentrifuge tube or mini-centrifuge strip well. Select the cuvette option in the NanoDrop OneC

software. Insert the cuvette noting the direction of the Micro assay (1:1 sample to reagent ratio): add light path indicated by the etched arrow. The optical 10 µL of working reagent to each of the standards and beam (2 mm) is directed 8.5 mm above the bottom sample tubes. of the cuvette. Refer to the cuvette manufacturer for

volume recommendations. After loading the cuvette Standard assay (1:30 sample to reagent ratio): with the appropriate volume, select Blank. add 300 µL of working reagent to each of the standard

and sample tubes. Note: The arm must be down for all measurements, 3. Add 10 µL of each standard and sample to each of except those made with cuvettes. It is recommended the reagent tubes. Mix well by gentle vortexing. If that cuvettes be removed from the instrument prior to necessary, collect the solution at the bottom of the making a pedestal measurement to ensure that the tube by a brief centrifugation. pedestal arm can move to the proper starting position.

4. It is advisable to use the dye reagent and protein buffer 5. Follow the direction at the top of the screen to measure (“0” reference) without any protein added as the zero the reference and standards. After each measurement, reference sample for this assay. wipe the upper and lower pedestals using a dry laboratory wipe. 5. Follow reagent manufacturer’s recommended incubation time. 6. After the last standard is measured, you can choose to load more standards or run samples using the window that appears in the lower right corner of the screen. To Protocol proceed with sample measurements, select the Run 1. Select the Proteins Tab from the New Experiment Samples radio button, and select DONE. Screen. Select the Protein Bradford application. 7. Enter a sample ID in the sample ID field located in the 2. On the left side of the screen, select the Curve Type upper left corner of the screen. Load 2 µL of sample and number of replicates to measure using the drop when using the pedestal. Select Measure. down menus. The Pierce protocol recommends using a 2nd order polynomial. Please note, the curve type 8. After completing all standard and sample cannot be changed after the assay is in progress. measurements, it is good practice to re-condition the pedestals using PR-1. 3. Enter the values for each standard concentration in the table on the right. The software allows for the 9. It is not necessary to blank the instrument between the reference and up to 7 additional standards. The zero standard and the unknown sample measurements. reference and standards can be measured with up to 3 replicates. Select DONE. Note: A fresh aliquot of sample should be used for each measurement. Note: The minimum requirement for standard curve 10. Cleaning the instrument after a measurement: generation is the measurement of two standards or the

measurement of the zero reference and at least one Pedestal Option: Simply wipe the upper and lower standard. It is recommended that additional standards pedestals using a dry laboratory wipe and the be included as necessary to cover the expected assay instrument is ready to measure the next sample. concentration range.

Cuvette Option: Remove the cuvette, and clean according to the manufacturer’s recommendations.

 BSA A595 Std. % (µg/mL) (n=3) dev. CV 0 0.44 0.003 NA 125 0.593 0.028 4.7 250 0.666 0.007 1.1 500 1.037 0.013 1.2 750 1.289 0.018 1.4 1000 1.445 0.015 1.0 1500 1.683 0.017 1.0 2000 1.783 0.012 0.7

Typical absorbance values and standard curve for a standard assay using 1:30 sample to reagent ratio assay using the Pierce Coomassie Plus reagent.

For additional information regarding the Bradford assay and reagents, please refer to the Pierce Website: thermoscientific.com/pierce

Toll free (US & Canada): 877-724-7690 • Technical support: [email protected]

Find out more at thermofisher.com/nanodrop

For Research Use Only. Not for use in diagnostic procedures. © 2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Once printed this document is no longer controlled. T141 Rev 08 April 2020  PROTOCOLPROTOCOL — — T140 T140 NanoDropNanoDrop One/One One/OneC C No. T140 No. T140 ProtocolProtocol PROTOCOL NanoDrop One/OneC Spectrophotometers ProteinProtein Lowry Lowry Assay Assay Lowry protein assay

IntroductionIntroduction TheThe Bio-Rad Bio-Rad® DC® DC Protein Protein Assay Assay1 is1 isa colorimetrica colorimetric assayassay for for protein protein concentration concentration following following detergent detergent solubilization.solubilization. The The reaction reaction is issimilar similar to tothat that described described in in thethe “Lowry “Lowry Assay: Assay: Protein Protein by by Folin Folin Reaction” Reaction” by by Lowry Lowry et et al alin inthe the Journal Journal of ofBiological Biological Chemistry Chemistry, 1951., 1951.

TheThe assay assay is isbased based on on the the reaction reaction of ofprotein protein with with an an alkalinealkaline copper copper tartrate tartrate solution solution and and Folin Folin reagent reagent in in a two-stepa two-step process process leading leading to tocolor color development. development. TheThe first first step step is isthe the reaction reaction between between protein protein and and Supplies,Supplies, Materials Materials Materials & &Reagents Reagents coppercopper in inan an alkaline alkaline medium medium and and then then the the reduction reduction • •NanoDrop NanoDrop One/One One/OneC SpectrophotometerC Spectrophotometer of ofFolin Folin reagent reagent by by the the copper-treated copper-treated protein. protein. Color Color • 0.5• 0.5 – 2– µL2 µL pipettor pipettor tips, tips, low low retention retention tips tips developmentdevelopment is isdue due primarily primarily to tothe the amino amino acids acids tyrosine tyrosine • 0.5 – 2 μL pipettor tips, low retention tips recommended recommendedrecommended andand tryptophan, tryptophan, but but also also to toa lessera lesser extent extent by by cystine, cystine, • 50 μL pipettor tips, low retention tips recommended cysteine,cysteine, and and histidine. histidine. The The reaction reaction will will have have a bluea blue color color • 50• 50 µL µL pipettor pipettor tips, tips, low low retention retention tips tips recommended recommended withwith maximum maximum absorbance absorbance at at750 750 nm nm using using Thermo Thermo • 400 μL pipettor tips, low retention tips recommended • 400• 400 µL µL pipettor pipettor tips, tips, low low retention retention tips tips recommended recommended ScientificScientific™ NanoDrop™ NanoDrop™ One/One™ One/OneC MicrovolumeC Microvolume UV-Vis UV-Vis • Low lint laboratory wipes SpectrophotometerSpectrophotometer and and the the Protein Protein Lowry Lowry Applications. Applications. • Low• Low lint lint laboratory laboratory wipes wipes

™ • •0.6 0.6 mL mL micro-centrifuge micro-centrifuge tubes tubes (Fisherbrand (Fisherbrand™ catalog™ catalog DynamicDynamic Range Range numbernumber 05-408-120) 05-408-120) TheThe assay assay has has a rangea range of of0.2 0.2 to to2.0 2.0 mg/mL. mg/mL. • •DC DC Protein Protein Assay Assay (Bio-Rad (Bio-RadReagents product product Package, number number 500-0116) 500-0116) A Astandard standard curve curve should should be be prepared prepared each each time time the the Bio-Rad PN 5000116 • Pierce• Pierce pre-diluted pre-diluted BSA BSA standards standards (P/N (P/N 23208) 23208) or orother other assayassay is isperformed. performed. We We recommend recommend choosing choosing a seconda second •commercially Thermocommercially Scientific prepared prepared™ Pierce protein protein™ BSA standards standards Standard Pre-diluted orderorder polynomial polynomial curve curve type type for for the the standard standard curve. curve. Set, PN 23208 or other commercially prepared protein • PR-1• PR-1 Kit Kit standards • PBS• PBS (Thermo (Thermo Fisher Fisher P/N P/N 28372) 28372) • Thermo Scientific™ NanoDrop™ PR-1 Reconditioning Kit, PN CHEM-PR1-KIT

• Thermo Scientific™ BupH™ PBS packs, Thermo Fisher PN 28372

 AssayAssay Recommendations Recommendations ProtocolProtocol • •Measure Measure 2 2μL μL sample sample aliquots. aliquots. 1. 1. From From the the NanoDrop NanoDrop One/One One/OneC NewC New Experiment Experiment screen,screen, tap tap the the Proteins Proteins tab tab and and then then tap tap the the “Protein “Protein • •Making Making standard standard and and sample sample measurements measurements in in Lowry”Lowry” icon icon to to open open the the application. application. triplicatetriplicate is isgood good practice. practice.

• •Re-condition Re-condition pedestals pedestals with with PR-1 PR-1 upon upon completion completion 2. 2. Enter Enter the the mg/mL mg/mL concentration concentration values values for for each each ofof assay. assay. standardstandard in inthe the table table on on the the right right in inthe the image image below. below.

SampleSample Preparation Preparation TheThe sample sample preparation preparation outlined outlined below below is isfor for pedestalpedestal measurements measurements on on the the NanoDrop NanoDrop One/One One/OneC C Spectrophotometer.Spectrophotometer. Follow Follow the the manufacturer’s manufacturer’s protocol protocol forfor a astandard standard assay assay when when making making measurements measurements in ina acuvette. cuvette.

1. 1. Equilibrate Equilibrate all all reagents, reagents, unknowns unknowns and and protein protein standardsstandards to to room room temperature. temperature. Mix Mix thoroughly thoroughly but but gentlygently to to avoid avoid introducing introducing micro micro bubbles. bubbles.

2. 2. If unknownIf unknown samples samples contain contain detergent, detergent, prepare prepare a a workingworking Reagent Reagent A, A, by by adding adding 20 20 µL µL of of Reagent Reagent S Sto to 3.3. Enter Enter the the number number of of replicates replicates for for each each standard. standard. We We eacheach mL mL of of Reagent Reagent A Athat that will will be be required required for for the the run. run. recommendrecommend three three replicates replicates per per standard, standard, including including LabelLabel this this as as “Working “Working Reagent Reagent A” A” with with an an expiration expiration thethe reference reference standard. standard. datedate of of one one week week post post preparation. preparation. If unknownIf unknown samplessamples do do not not contain contain detergent, detergent, omit omit this this step step and and 4.4. Select Select the the curve curve type. type. We We recommend recommend a seconda second order order useuse Reagent Reagent A Aas as supplied supplied in inthe the kit. kit. polynomialpolynomial curve curve type type for for Protein Protein Lowry Lowry Protein Protein Assay. Assay.

3.3. Prepare Prepare standards standards to to cover cover the the range range of of the the assay assay (0.2(0.2 to to 2.0 2.0 mg/mL). mg/mL). A Astandard standard curve curve should should be be preparedprepared each each time time unknown unknown samples samples will will be be tested. tested. If notIf not using using pre-diluted pre-diluted standards, standards, for for best best results, results, the the standardsstandards should should be be diluted diluted in inthe the same same buffer buffer as as the the unknownunknown samples. samples.

4.4. Label Label all all tubes tubes and and pipet pipet 10 10 µL µL of of standards standards and and unknownunknown samples samples into into appropriately appropriately labeled labeled micro- micro- centrifugecentrifuge tubes. tubes. Pipet Pipet 10 10 µL µL of of PBS PBS into into the the reference reference oror zero zero standard. standard.

5.5. Add Add 50 50 µL µL of of either either Working Working Reagent Reagent A Aor or Reagent Reagent A A 5.5. Press Press Done Done when when complete. complete. suppliedsupplied in inthe the kit kit (see (see step step #2) #2) to to each each tube. tube. Mix Mix well. well. 6.6. At At the the prompt, prompt, clean clean both both pedestals pedestals and and make make a blanka blank 6.6. Add Add 400 400 µL µL of of Reagent Reagent B Bto to each each tube. tube. Mix Mix well, well, but but measurementmeasurement using using water water as as the the blanking blanking solution. solution. notnot so so vigorously vigorously as as to to introduce introduce micro-bubbles micro-bubbles into into thethe tubes. tubes. 7. 7. Follow Follow the the on-screen on-screen prompts prompts to to measure measure each each replicatereplicate of of each each standard, standard, taking taking care care to to wipe wipe 7. 7. Incubate Incubate all all tubes tubes at at room room temperature temperature for for 15 15 minutes. minutes. thethe upper upper and and lower lower pedestal pedestal between between each each measurement.measurement. Each Each replicate replicate measurement measurement of of the the referencereference or or standards standards should should be be made made using using a fresha fresh aliquotaliquot on on the the pedestal. pedestal.

 8.8. You You will will notice notice in inthe the upper upper right right corner corner of of the the screen screen StandardStandard Curve Curve Data Data thethe note note in inred: red: Invalid Invalid Curve Curve. When. When enough enough data data BSABSA A(595)A(595) Std.Std. dev. dev. %CV%CV pointspoints have have been been measured measured to to plot plot the the curve curve type type (µ(g/mL)µg/mL) (n=3)(n=3) selected,selected, this this now now will will change change from from red red to to blue: blue: Valid Valid 0 0 0.440.44 0.0030.003 NANA CurveCurve. For. For a seconda second order order polynomial polynomial curve, curve, at at least least 125125 0.5930.593 0.0280.028 4.74.7 threethree standards, standards, including including the the reference reference must must be be 250250 0.6660.666 0.0070.007 1.11.1 measuredmeasured before before a curvea curve can can be be constructed. constructed. This This 500500 1.0371.037 0.0130.013 1.21.2 indicationindication does does not not address address the the quality quality of of the the curve, curve, 750750 1.2891.289 0.0180.018 1.41.4 it isit isa notificationa notification that that enough enough standards standards have have been been 10001000 1.4451.445 0.0150.015 1.01.0 measuredmeasured to to construct construct the the curve curve type type selected. selected. 15001500 1.6831.683 0.0170.017 1.01.0 20002000 1.7831.783 0.0120.012 0.70.7 9.9. When When the the last last replicate replicate of of the the last last standard standard has has been been readread the the following following message message will will appear: appear:

10.10. At At this this point, point, you you may may choose choose to to run run more more standards standards oror begin begin running running samples. samples. Please Please note note that that once once samplessamples are are measured, measured, you you will will not not be be able able to to TheThe above above data data is istypical typical of of absorbance absorbance values values and and makemake changes changes to to the the standard standard curve curve by by measuring measuring standardstandard curve curve using using the the DC DC Protein Protein Assay. Assay. additionaladditional standards. standards. ForFor additional additional information information regarding regarding the the Lowry Lowry assay assay 11.11. It isIt isnot not necessary necessary to to blank blank the the instrument instrument andand reagents, reagents, please please refer refer to to the the manufacturer’s manufacturer’s product product betweenbetween measurement measurement of of the the standards standards and and the the literatureliterature supplied supplied with with the the DC DC Protein Protein Assay. Assay. unknownunknown samples. samples. ReferencesReferences 1. 1. DC DC Protein Protein Assay Assay Instruction Instruction Manual, Manual, LIT449 LIT449 Rev. Rev. D Bio-RadD Bio-Rad Laboratories. Laboratories. 12.12. After After each each measurement, measurement, clean clean the the upper upper and and lower lower pedestalspedestals with with a clean,a clean, dry dry laboratory laboratory wipe. wipe.

13.13. Upon Upon completion completion of of all all measurements, measurements, we we recommendrecommend cleaning cleaning and and reconditioning reconditioning the the measurementmeasurement pedestals pedestals using using PR-1. PR-1.

InIn the the United United States: States: InIn Canada: Canada: ForFor customer customer service, service, call call 1-800-766-7000 1-800-766-7000 ForFor customer customer service, service, call call 1-800-234-7437 1-800-234-7437 ToTo fax fax an an order, order, use use 1-800-926-1166 1-800-926-1166 ToTo fax fax an an order, order, use use 1-800-463-2996 1-800-463-2996 TollTo freeTo order (US order & Canada): online: online: 877-724-7690 thermofisher.com thermofisher.com • Technical support: [email protected] order order online: online: thermofisher.ca thermofisher.ca

Find FindFind outout out moremore more atat at thermofisher.com/nanodropthermofisher.com/nanodrop thermofisher.com/nanodrop

For Research Use Only. Not for use in diagnostic procedures. © 2020 Thermo Fisher Scientific Inc. All rights reserved. Bio-Rad is a registered trademark of Bio-Rad Laboratories, Inc. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise© 2018© 2018 Thermospecified. Thermo Fisher Once Fisher printedScientific Scientific this document Inc. Inc. Bio-Rad isBio-Rad no longer is isa registeredcontrolled.a registered T140 trademark trademark Rev 07 of April ofBio-Rad Bio-Rad 2020 Laboratories. Laboratories. All Allother other trademarks trademarks areare the the property property of Thermoof Thermo Fisher Fisher Scientific Scientific and and its itssubsidiaries subsidiaries unless unless otherwise otherwise specified. specified. T140 T140 Rev. Rev. 07 07Aug Aug 2018 2018  PROTOCOL NanoDrop One/OneC Spectrophotometers

Pierce 660 nm protein assay

Introduction Supplies The Thermo Scientific™ Pierce™ 660 nm Protein Assay reagent Equipment: is a ready-to-use formulation that offers rapid, accurate • NanoDrop One/OneC Spectrophotometer and reproducible colorimetric detection of minute amounts of protein in solution. Used in conjunction with the micro- • 0.5-2 µL pipettor (and low retention tips) and 10-1000 µL volume capability of the Thermo Scientific™ NanoDrop™ pipettors Spectrophotometers, the reagent provides an accurate and rapid means of protein quantitation with minimal consumption Materials: of sample. The ability of NanoDrop Spectrophotometers • Low lint laboratory wipes to measure as little as 2 µL of protein samples allows significantly scaled-down reaction volumes, thereby using • 0.5 mL microcentrifuge tubes or 0.2 mL mini-centrifuge only a fraction of sample and reagent commonly needed for strip tubes and caps conventional cuvette-based instruments. Reagents: Note: All specifications and protocol instructions presented • Thermo Scientific™ Pierce™ 660 nm Protien Assay below are specific to the pedestal mode for NanoDrop Reagent, Pierce PN 22660 One/OneC instruments. Please refer to the reagent ™ ™ manufacturer for additional guidance when utilizing the • Thermo Scientific Pierce BSA Pre-Diluted Standards cuvette mode of the NanoDrop OneC. Pre-diluted Set, PN 23208 or other commercially prepared protein standard

Dynamic range • Thermo Scientific™ NanoDrop™ PR-1 Reconditioning Kit, The assay has a linear range of 50–2000 µg/mL using a PN Chem-PR1-KIT 1:15 sample to reagent ratio. The sensitivity of the assay may be increased by using a 1:7.5 sample to reagent ratio Assay recommendations yielding a linear range of 25–1000 µg/mL. • Measure 2 µL sample aliquots.

• Re-condition pedestals with PR-1 upon assay completion.

Thermo Scientific NanoDrop Onec Microvolume UV-Vis Spectrophotometer

 Sample preparation 3. In the table on right side of the screen, enter the values 1. Equilibrate all reagents, unknowns and protein for each standard concentration. The software allows standards to room temperature. Mix thoroughly but for the reference and up to 7 additional standards. The gently to avoid introducing micro bubbles. zero reference and standards can be measured with up to 3 replicates. Tap Done. 2. Prepare a zero reference (0 mg/mL protein).

For a 1:15 sample to working reagent ratio: Add 10 µL of the assay buffer to 150 µL of the Pierce 660 reagent

For a 1:7.5 sample to working reagent ratio: Add 20 µL of the assay buffer to 150 µl of the Pierce 660 reagent.

Note: The zero reference solution is used as the ‘blank’. This is unlike the other colorimetric assays run on NanoDrop instruments where water is used for the ‘blank’ measurement. Note: The minimum requirement for standard curve 3. Prepare standards and samples. generation is the measurement of the zero reference and at least one standard. It is recommended that For a 1:15 sample to working reagent ratio: Add enough standards be included to cover the expected 10 µL of each standard and sample to 150 µL of the assay concentration range. Pierce 660 reagent. 4. Establish a blank using the appropriate buffer. It is For a 1:7.5 sample to working reagent ratio: 20 µL advisable to use the dye reagent and protein buffer (“0” of each standard and sample to 150 µL of the Pierce reference) without any protein added as both the blank 660 reagent. and zero reference sample for this assay. 4. Mix each standard and unknown sample thoroughly by gently pipetting up and down several times. If Pedestal Option: Pipette 2 µl of blank solution onto the necessary, collect the solution at the bottom of the bottom pedestal, lower the arm and tap Blank. tube by a brief centrifugation. Cuvette Option: (Model NanoDrop OneC only): Insert 5. Incubate at room temperature for 5 minutes. the cuvette noting the direction of the light path indicated by the etched arrow. The optical beam (2 mm) is directed 8.5 mm above the bottom of the Protocol cuvette. Refer to the cuvette manufacturer for volume 1. Tap the Protein tab from the Home screen. Tap the recommendations. Protein Pierce 660 nm application button.

2. On the left side of the screen, select the Curve Type Note: It is recommended that cuvettes be removed and number of replicates to measure. We recommend from the instrument prior to making a pedestal selecting the Linear curve type and measuring measurement to ensure that the pedestal arm can 3 replicates of each standard. move to the proper starting position.

5. A message at the top of the screen will prompt you Optional: The user has the option to measure to, “Clean both pedestals and Load – Reference the absorbance of each standard or enter the 0.000,” if you are using the pedestal mode. If you are manufacturer supplied standard absorbance values using the cuvette mode, a message will prompt you manually. In the bottom left pane, select the box to to, “Insert Cuvette and Load - Reference 0.000.” enter the manufacture supplied standard absorbance Load the reference sample in the manner described for values manually. Deselect the box to measure the loading the blank. absorbance of each standard. See image below.

 1. After each measurement, wipe the upper and lower 2. Swipe the screen to the left to view the curve. pedestals using a clean, dry laboratory wipe.

2. Once the reference and at least one standard have been measured, the message Invalid Curve in red at the top right of the screen will switch to Valid Curve in blue.

3. After all standard measurements have been made a pop-up box will indicate Standards Complete. Select Load more standards to enter and measure additional standards or Run samples if standard measurements are complete.

4. After selecting Run samples, enter a sample ID at the top of the screen. Load 2 µL of sample when using the pedestal. Tap Measure. 3. Swipe the screen again to the left to view your measurement results. 5. It is not necessary to blank the instrument between the standard and the unknown sample measurements.

Note: A fresh aliquot of sample should be used for each measurement.

6. After completing all Standard and Sample measurements, it is good practice to re-condition the pedestals using PR-1.

After the measurements: 1. Select each sample to display multiple spectra at a time.

Standard curve data

BSA A660 Std. % (µg/mL) (n=3) dev. CV 0 0.072 0.003 NA 125 0.223 0.004 1.6 250 0.403 0.002 0.5 500 0.757 0.013 1.7 750 1.107 0.002 0.2 1000 1.467 0.006 0.4 1500 2.065 0.012 0.6 2000 2.676 0.074 2.8

Table 1. Typical absorbance values for a 1:15 sample to reagent ratio assay using the Pierce 660 nm Protein Assay.

For additional information regarding the Pierce 660 nm assay and reagents: thermoscientific.com/pierce

Toll free (US & Canada): 877-724-7690 • Technical support: [email protected]

Find out more at thermofisher.com/nanodrop

For Research Use Only. Not for use in diagnostic procedures. © 2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Once printed this document is no longer controlled. T139 Rev 01 April 2020  APPLICATIONPROTOCOLAPPLICATION NOTE NOTE NanoDropNanoDropNanoDrop One/One One/One One/OneC C C No. T144 No. T144 No. A AA customcustom custom methodmethod method forfor for hemoglobinhemoglobin measurements measurements UsingUsing the thethe NanoDrop NanoDrop NanoDrop One One Spectrophotometer OneSpectrophotometer Spectrophotometer

AbstractAbstract ThermoThermo Scientific Scientific™ NanoDrop™ NanoDrop™ Microvolume™ Microvolume UV-Vis UV-Vis SpectrophotometersSpectrophotometers have have been been used used for for many many years years to toreliably reliably measure measure nucleic nucleic acids acids and and proteins proteins using using as aslittle little as as1–2 1–2 µL µL of ofsample. sample. However, However, NanoDrop NanoDrop spectrophotometersspectrophotometers may may also also be be used used for for many many other other applications.applications. This This application application note note discusses discusses how how the the NanoDropNanoDrop One/One One/OneC SpectrophotometerC Spectrophotometer can can be be used used to to measuremeasure hemoglobin hemoglobin using using a customa custom method. method.

IntroductionIntroduction HemoglobinHemoglobin is ais tetramerica tetrameric protein protein found found in erythrocytesin erythrocytes thatthat transports transports oxygen oxygen to totissues tissues throughout throughout the the body body andand carries carries CO CO2 away2 away from from tissues tissues [1]. [1]. Hemoglobin Hemoglobin can can reversiblyreversibly bind bind oxygen oxygen and and typically typically contains contains iron iron in thein the reducedreduced ferrous ferrous state state (Fe (Fe2+).2+ When). When the the iron iron is oxidizedis oxidized to tothe the ferric ferric state state (Fe (Fe3+),3+ hemoglobin), hemoglobin is convertedis converted to to methemoglobinmethemoglobin and and can can no no longer longer bind bind oxygen oxygen [2]. [2].

A spectrophotometerA spectrophotometer may may be be used used to toquantitate quantitate various various formsforms of ofhemoglobin hemoglobin using using the the appropriate appropriate extinction extinction coefficientcoefficient and and peak peak absorbance absorbance value. value. Oxyhemoglobin Oxyhemoglobin containscontains bound bound oxygen oxygen and and exhibits exhibits absorbance absorbance peaks peaks at at414 414 nm, nm, 541 541 nm, nm, and and 576 576 nm nm [3]. [3]. Deoxyhemoglobin Deoxyhemoglobin A spectrophotometerA spectrophotometer may may also also be be used used to toassess assess a a doesdoes not not contain contain bound bound oxygen oxygen and and exhibits exhibits an an plasmaplasma or orserum serum sample sample for for hemolysis. hemolysis. Hemolysis Hemolysis occurs occurs absorbanceabsorbance peak peak at at431 431 nm. nm. Methemoglobin Methemoglobin cannot cannot whenwhen the the red red blood blood cell cell membrane membrane breaks breaks down down and and bindbind oxygen oxygen due due to tocontaining containing iron iron in thein the ferric ferric state, state, and and releasesreleases hemoglobin hemoglobin and and other other intracellular intracellular components components exhibitsexhibits an anabsorbance absorbance peak peak at at406 406 nm nm [4]. [4]. intointo the the surrounding surrounding serum serum or orplasma plasma [5]. [5]. The The estimation estimation

 of ofhemolysis hemolysis is importantis important as asit can it can affect affect the the accuracy accuracy peakspeaks at atthese these wavelengths wavelengths [5]. [5]. The The method method includes includes of ofmany many laboratory laboratory assays. assays. A spectrophotometerA spectrophotometer may may wavelengthwavelength 406 406 nm nm to tomonitor monitor for for methemoglobin. methemoglobin. A A bebe used used to toevaluate evaluate hemolysis hemolysis using using the the absorbance absorbance baselinebaseline correction correction at at750 750 nm nm was was included included for for the the of ofa plasmaa plasma or orserum serum sample sample at at414 414 nm. nm. An An elevated elevated entireentire spectrum, spectrum, as aswell well as asan ananalysis analysis wavelength wavelength absorbanceabsorbance at at414 414 nm nm is associatedis associated with with increased increased free free correctioncorrection based based on on the the sloping sloping baseline baseline correction correction from from oxyhemoglobinoxyhemoglobin [3, [3,6]. 6]. 360–500360–500 nm. nm.

It isIt importantis important to tokeep keep in mindin mind that that spectrophotometers spectrophotometers TheThe hemoglobin hemoglobin custom custom method method was was validated validated using using measuremeasure the the total total absorbance absorbance of ofa sample,a sample, and and hemoglobinhemoglobin obtained obtained from from MP MP Biomedicals, Biomedicals, LLC LLC (catalog (catalog cannotcannot distinguish distinguish the the analyte analyte of ofinterest interest from from any any numbernumber 100714, 100714, lot lotnumber number 7541J). 7541J). The The hemoglobin hemoglobin otherother component component in thein the sample sample that that absorbs absorbs at atthe the preparationspreparations available available through through MP MP Biomedicals Biomedicals may may be be samesame wavelength. wavelength. Spectrophotometric Spectrophotometric hemoglobin hemoglobin primarilyprimarily methemoglobin, methemoglobin, which which cannot cannot bind bind oxygen, oxygen, as as measurementsmeasurements in plasmain plasma may may be be subject subject to tointerference interference thethe iron iron in hemoglobinin hemoglobin is easilyis easily oxidized oxidized by byair. air. fromfrom increased increased bilirubin bilirubin levels, levels, plasma plasma proteins, proteins, albumin, albumin, lipids,lipids, and and other other absorbing absorbing components components [5]. [5]. A hemoglobinA hemoglobin stock stock was was prepared prepared at at8 mg/mL8 mg/mL usingusing reagent reagent grade grade water. water. Serial Serial dilutions dilutions were were If youIf you would would like like to toquantitate quantitate hemoglobin hemoglobin in wholein whole thenthen performed performed to tocreate create standards standards at at4 mg/mL,4 mg/mL, blood,blood, a colorimetrica colorimetric assay assay may may be be performed performed at at540 540 nm nm 2 mg/mL,2 mg/mL, 1 mg/mL,1 mg/mL, 0.5 0.5 mg/mL, mg/mL, 0.25 0.25 mg/mL, mg/mL, and and usingusing Drabkin’s Drabkin’s reagent. reagent. A colorimetricA colorimetric assay assay is beyondis beyond 0.1250.125 mg/mL. mg/mL. The The instrument instrument was was blanked blanked with with water, water, thethe scope scope of ofthis this application application note, note, but but users users can can set set andand five five replicates replicates of eachof each standard standard were were measured measured upup a customa custom method method for for the the assay assay on on the the NanoDrop NanoDrop usingusing the the Oxy-hemoglobin Oxy-hemoglobin Custom Custom Method Method on on the the One/OneOne/OneC Spectrophotometer.C Spectrophotometer. NanoDropNanoDrop One/One One/OneC Spectrophotometer.C Spectrophotometer.

ExperimentalExperimental Procedures Procedures ResultsResults A customA custom method method was was created created for for the the NanoDrop NanoDrop EachEach hemoglobin hemoglobin standard standard was was measured measured in replicatesin replicates One/OneOne/OneC instrumentC instrument to tomeasure measure the the absorbance absorbance of of of offive five on onthe the NanoDrop NanoDrop One One instrument instrument using using the the hemoglobinhemoglobin at at406 406 nm, nm, 414 414 nm, nm, 431 431 nm, nm, 541 541 nm, nm, and and hemoglobinhemoglobin custom custom method. method. The The standard standard deviation deviation 576576 nm. nm. The The method method includes includes an ananalysis analysis wavelength wavelength at at basedbased on on five five replicates replicates of eachof each solution solution ranged ranged from from 414414 nm nm to tocalculate calculate the the concentration concentration of ofoxyhemoglobin oxyhemoglobin 0.015–0.552A.0.015–0.552A. The The coefficient coefficient of variationof variation for for each each usingusing an anextinction extinction coefficient coefficient of of524,280 524,280 M-1 cmM-1 cm-1 -1 solutionsolution ranged ranged from from 0.61–2.79%. 0.61–2.79%. andand molecular molecular weight weight of of64.5 64.5 kDa kDa [7]. [7]. The The method method alsoalso includes includes an anadditional additional calculation calculation to todetermine determine AverageAverage StandardStandard A406A406 DeviationDeviation %CV%CV thethe concentration concentration of ofdeoxyhemoglobin deoxyhemoglobin using using the the n=5n=5 n=5n=5 absorbanceabsorbance at at431 431 nm, nm, an anextinction extinction coefficient coefficient of of SolutionSolution 1 1 44.49344.493 0.4870.487 1.091.09 552,160552,160 M -1M cm-1 cm-1, and-1, and molecular molecular weight weight of of64.5 64.5 kDa kDa [7]. [7]. SolutionSolution 2 2 22.84622.846 0.5520.552 2.412.41 TheThe method method includes includes wavelengths wavelengths 541 541 nm nm and and 576 576 nm nm to tomonitor, monitor, as asoxyhemoglobin oxyhemoglobin exhibits exhibits absorbance absorbance SolutionSolution 3 3 11.12711.127 0.0680.068 0.610.61 SolutionSolution 4 4 5.5495.549 0.0580.058 1.051.05 SolutionSolution 5 5 2.6602.660 0.0190.019 0.710.71 CustomCustom Method Method Download Download SolutionSolution 6 6 1.2451.245 0.0350.035 2.792.79 1. Navigate1. Navigate to www.thermofisher.com/nanodropto www.thermofisher.com/nanodrop SolutionSolution 7 7 0.5790.579 0.0150.015 2.662.66 2. On2. Onthe theleft, left, select select “NanoDrop “NanoDrop Software Software Download” Download” TableTable 1. Results 1. Results for forhemoglobin hemoglobin standards standards measured measured using using the the 3. Choose3. Choose the the“NanoDrop “NanoDrop One/One One/OneC” tabC” tab hemoglobinhemoglobin custom custom method method on onthe the NanoDrop NanoDrop One/One One/OneC. C. 4. Select4. Select “Local “Local Control Control Software Software Download Download Instructions” Instructions” 5. Scroll5. Scroll to “Howto “How to addto add a NanoDrop a NanoDrop One/One One/OneC CustomC Custom Method Method ConclusionConclusion file”file” and and click click on “Oxy-hemoglobinon “Oxy-hemoglobin Method” Method” TheThe hemoglobin hemoglobin custom custom method method described described in in 6. Unzip6. Unzip the thecustom custom method method file file and and copy copy the the.method .method file fileto ato USBa USB thisthis application application note note can can be be used used to todetermine determine devicedevice and and then then follow follow the theonline online “Instructions “Instructions for foruploading uploading a a oxyhemoglobinoxyhemoglobin and and deoxyhemoglobin deoxyhemoglobin concentrations concentrations on on CustomCustom Method Method to theto theinstrument instrument from from a USB a USB device”. device”. thethe NanoDrop NanoDrop One/One One/OneC Spectrophotometer.C Spectrophotometer. Depending Depending onon your your needs, needs, the the method method may may be be used used to tomonitor monitor the the

 Figure 1. Typical absorbance spectrum for methemoglobin measured on the NanoDrop One with a peak at 406 nm. The spectra also lacks peaks at 541 nm and 576 nm which are present in oxyhemoglobin.

6. J. S. Shah, P. S. Soon, D. J. Marsh, Comparison of Methodologies to Detect Low absorbance at 414 nm to determine if the sample may Levels of Hemolysis in Serum for Accurate Assessment of Serum microRNAs. PLOS be hemolyzed. If measuring oxyhemoglobin, the method ONE. 11, e0153200 (2016). may be used to monitor the absorbance at 541 nm and 7. Optical Absorption of Hemoglobin, (available at http://omlc.org/spectra/hemoglobin/ index.html). 576 nm. If measuring methemoglobin, the method also may be used to monitor the absorbance at 406 nm. Further Assistance and Technical Support For further assistance, contact NanoDrop technical References support at [email protected] or visit 1. Peter J. Kennelly, PhD & Victor W. Rodwell, PhD, in Harper’s Illustrated Biochemistry (McGraw-Hill Companies, Inc, 29th Edition., 2012), pp. 48–55. thermofisher.com/nanodrop. 2. Trefor Higgins, M.Sc., John H. Eckfeldt, M.D., Ph.D., James C. Barton, M.D., Basil T. Doumas, Ph.D., in Tietz Textbook of Clinical Chemistry and Molecular Diagnostics (Elsevier Saunders, Fifth Edition., 2012), pp. 985–988. 3. M. B. Kirschner et al., Haemolysis during Sample Preparation Alters microRNA Content of Plasma. PLOS ONE. 6, e24145 (2011). 4. E. J. van Kampen, W. G. Zijlstra, in Advances in Clinical Chemistry, A. L. Latner, M. Figure 1. Typical absorbance spectrum for methemoglobin measured on theK. Schwartz, NanoDrop Eds. (Elsevier, 1983; http://www.sciencedirect.com/science/article/pii/ One with a peak at 406 nm. The spectra also lacks peaks at 541 nm and 576 S0065242308604011),nm which are vol. 23, pp. 199–257. present in oxyhemoglobin. 5. S. O. Sowemimo-Coker, Red blood cell hemolysis during processing. Transfus. Med. Rev. 16, 46–60 (2002). 6. J. S. Shah, P. S. Soon, D. J. Marsh, Comparison of Methodologies to Detect Low absorbance at 414 nm to determine if the sample may Levels of Hemolysis in Serum for Accurate Assessment of Serum microRNAs. PLOS be hemolyzed. If measuring oxyhemoglobin, the method ONE. 11, e0153200 (2016). may be used to monitor the absorbance at 541 nm and In7. Opticalthe United Absorption States: of Hemoglobin, (available at http://omlc.org/spectra/hemoglobin/In Canada: index.html). 576 nm. If measuring methemoglobin, the method also For customer service, call 1-800-766-7000 For customer service, call 1-800-234-7437 may be used to monitor the absorbance at 406 nm. ToFurther fax an order,Assistance use 1-800-926-1166 and Technical SupportTo fax an order, use 1-800-463-2996 To order online: thermofisher.com To order online: thermofisher.ca TollFor free (USfurther & Canada): assistance,877-724-7690 • Technical contact support: [email protected] NanoDrop technical References support at [email protected] or visit 1. Peter J. Kennelly, PhD & Victor W. Rodwell, PhD, in Harper’s Illustrated Biochemistry thermofisher.com/nanodrop. (McGraw-Hill Companies, Inc, 29th Edition., 2012), pp. 48–55. Find Find outout moremore atat thermofisher.com/nanodropthermofisher.com/nanodrop 2. Trefor Higgins, M.Sc., John H. Eckfeldt, M.D., Ph.D., James C. Barton, M.D., Basil T. Doumas, Ph.D., in Tietz Textbook of Clinical Chemistry and Molecular Diagnostics For Research Use Only. Not for use in diagnostic procedures. © 2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks (Elsevier Saunders, Fifth Edition., 2012), pp. 985–988. are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Once printed this document is no longer controlled. T144© 2018 Rev Thermo 29 April Fisher 2020 Scientific Inc. All other trademarks are the property of Thermo Fisher Scientific and its 3. M. B. Kirschner et al., Haemolysis during Sample Preparation Alters microRNA subsidiaries unless otherwise specified.T144 Rev. 29 Mar 2018 Content of Plasma. PLOS ONE. 6, e24145 (2011).  4. E. J. van Kampen, W. G. Zijlstra, in Advances in Clinical Chemistry, A. L. Latner, M. K. Schwartz, Eds. (Elsevier, 1983; http://www.sciencedirect.com/science/article/pii/ S0065242308604011), vol. 23, pp. 199–257. 5. S. O. Sowemimo-Coker, Red blood cell hemolysis during processing. Transfus. Med. Rev. 16, 46–60 (2002).

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