Western Blotting Guidebook
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Supplementary Materials and Method Immunostaining and Western Blot
Supplementary Materials and Method Immunostaining and Western Blot Analysis For immunofluorescence staining, mouse and human cells were fixed with 4% paraformaldehyde- PBS for 15 min. Following Triton-X100 permeabilization and blocking, cells were incubated with primary antibodies overnight at 4°C following with Alexa 594-conjugated secondary antibodies at 4°C for 1 hour (Thermo Fisher Scientific, 1:1000). Samples were mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories) and immunofluorescence was detected using Olympus confocal microscopy. For western blot analysis, cells were lysed on ice using RIPA buffer supplemented with protease and phosphatase inhibitors (Sigma). Primary Antibodies for Immunostaining and Western Blot Analysis: Yap (14074, Cell Signaling), pYAP (4911, Cell Signaling), Lats1 (3477, Cell Signaling), pLats1( 8654, Cell Signaling), Wnt5a (2530, Cell Signaling), cleaved Caspase-3 (9661, Cell Signaling), Ki-67 (VP-K451, Vector Laboratories), Cyr61 (sc-13100, Santa Cruz Biotechnology), CTGF (sc-14939, Santa Cruz Biotechnology), AXL (8661, Cell Signaling), pErk (4376, Cell Signaling), pMEK (4376, Cell Signaling), Ck-19 (16858-1-AP, Proteintech), Actin (A2228, Sigma Aldrich), Vinculin (V4139, Sigma Aldrich), Kras (sc-30, Santa Cruz Biotechnology). Ectopic expression of YAP1 and WNT5A in mouse and human cells To generate YAP1S127A-expressing stable Pa04C cells, Pa04C cells were transfected with a linearized pcDNA3.1 plasmid with or without YAP1 cDNA containing S127A substitution. Two days post-transfection using Lipofectamine1000, cultures were selected in G418 (Sigma) and single clones were picked and expanded for further analysis. Overexpression of YAPS127A or WNT5A in human or mouse cells other than Pa04C were acheieved with lentivral infection. Briefly, lentivirus infection was performed by transfecting 293T cells with either GFP control, YAP1S127A, or WNT5A cloned in pHAGE lentivirus vector {EF1α promoter-GW-IRES-eGFP (GW: Gateway modified)}. -
Agarose Gel Electrophoresis
Laboratory for Environmental Pathogen Research Department of Environmental Sciences University of Toledo Agarose gel electrophoresis Background information Agarose gel electrophoresis of DNA is used to determine the presence and distinguish the type of nucleic acids obtained after extraction and to analyze restriction digestion products. Desired DNA fragments can be physically isolated for various purposes such as sequencing, probe preparation, or for cloning fragments into other vectors. Both agarose and polyacrylamide gels are used for DNA analysis. Agarose gels are usually run to size larger fragments (greater than 200 bp) and polyacrylamide gels are run to size fragments less than 200 bp. Typically agarose gels are used for most purposes and polyacrylamide gels are used when small fragments, such as digests of 16S rRNA genes, are being distinguished. There are also specialty agaroses made by FMC (e.g., Metaphor) for separating small fragments. Regular agarose gels may range in concentration from 0.6 to 3.0%. Pouring gels at less or greater than these percentages presents handling problems (e.g., 0.4% agarose for genomic DNA partial digests requires a layer of supporting 0.8% gel). For normal samples make agarose gels at 0.7%. The chart below illustrates the optimal concentrations for fragment size separation. The values listed are approximate and can vary depending on the reference that is used. If you do not know your fragment sizes then the best approach is to start with a 0.7% gel and change subsequently if the desired separation is not achieved. Nucleic acids must be stained prior to visualization. Most laboratories use ethidium bromide but other stains (e.g., SYBR green, GelStar) are available. -
Green Fluorescent Protein (GFP) Purification Student Manual
Green Fluorescent Protein (GFP) Purification Student Manual "Bioengineered DNA was, weight for weight, the most valuable material in the world. A single microscopic bacterium, too small to see with the human eye, but containing the gene for a heart attack enzyme, streptokinase, or for "ice-minus" which prevented frost damage to crops, might be worth 5 billion dollars to the right buyer." Michael Crichton - Jurassic Park Contents Lesson 1 Genetic Transformation Review—Finding the Green Fluorescent Molecule Lesson 2 Inoculation—Growing a Cell Culture Lesson 3 Purification Phase 1—Bacterial Concentration and Lysis Lesson 4 Purification Phase 2—Removing Bacterial Debris Lesson 5 Purification Phase 3—Protein Chromatography 26 Lesson 1 Finding the Green Fluorescent Molecule Genetic Transformation Review In Bio-Rad Kit 1, you performed a genetic transformation of E. coli bacterial cells. The results of this procedure were colonies of cells that fluoresced when exposed to ultraviolet light. This is not a normal phenotype (characteristic) for E.coli. You were then asked to fig- ure out a way to determine which molecule was becoming fluorescent under UV light. After determining that the pGLO plasmid DNA was not responsible for the fluorescence under the UV light, you concluded that it was not the plasmid DNA that was fluorescing in response to the ultraviolet light within the cells. This then led to the next hypothesis that if it is not the DNA fluorescing when exposed to the UV light, then it must be a protein that the new DNA pro- duces within the cells. 1. Proteins. a. What is a protein? b. -
Protocols and Tips in Protein Purification
Department of Molecular Biology & Biotechnology Protocols and tips in protein purification or How to purify protein in one day Second edition 2018 2 Contents I. Introduction 7 II. General sequence of protein purification procedures 9 Preparation of equipment and reagents 9 Preparation and use of stock solutions 10 Chromatography system 11 Preparation of chromatographic columns 13 Preparation of crude extract (cell free extract or soluble proteins fraction) 17 Pre chromatographic steps 18 Chromatographic steps 18 Sequence of operations during IEC and HIC 18 Ion exchange chromatography (IEC) 19 Hydrophobic interaction chromatography (HIC) 21 Gel filtration (SEC) 22 Affinity chromatography 24 Purification of His-tagged proteins 25 Purification of GST-tagged proteins 26 Purification of MBP-tagged proteins 26 Low affinity chromatography 26 III. “Common sense” strategy in protein purification 27 General principles and tips in “common sense” strategy 27 Algorithm for development of purification protocol for soluble over expressed protein 29 Brief scheme of purification of soluble protein 36 Timing for refined purification protocol of soluble over -expressed protein 37 DNA-binding proteins 38 IV. Protocols 41 1. Preparation of the stock solutions 41 2. Quick and effective cell disruption and preparation of the cell free extract 42 3. Protamin sulphate (PS) treatment 43 4. Analytical ammonium sulphate cut (AM cut) 43 5. Preparative ammonium sulphate cut 43 6. Precipitation of proteins by ammonium sulphate 44 7. Recovery of protein from the ammonium sulphate precipitate 44 8. Analysis of solubility of expression 45 9. Analysis of expression for low expressed His tagged protein 46 10. Bio-Rad protein assay Sveta’s easy protocol 47 11. -
Blotting Techniques Blotting Is the Technique in Which Nucleic Acids Or
Blotting techniques Blotting is the technique in which nucleic acids or proteins are immobilized onto a solid support generally nylon or nitrocellulose membranes. Blotting of nucleic acid is the central technique for hybridization studies. Nucleic acid labeling and hybridization on membranes have formed the basis for a range of experimental techniques involving understanding of gene expression, organization, etc. Identifying and measuring specific proteins in complex biological mixtures, such as blood, have long been important goals in scientific and diagnostic practice. More recently the identification of abnormal genes in genomic DNA has become increasingly important in clinical research and genetic counseling. Blotting techniques are used to identify unique proteins and nucleic acid sequences. They have been developed to be highly specific and sensitive and have become important tools in both molecular biology and clinical research. General principle The blotting methods are fairly simple and usually consist of four separate steps: electrophoretic separation of protein or of nucleic acid fragments in the sample; transfer to and immobilization on paper support; binding of analytical probe to target molecule on paper; and visualization of bound probe. Molecules in a sample are first separated by electrophoresis and then transferred on to an easily handled support medium or membrane. This immobilizes the protein or DNA fragments, provides a faithful replica of the original separation, and facilitates subsequent biochemical analysis. After being transferred to the support medium the immobilized protein or nucleic acid fragment is localized by the use of probes, such as antibodies or DNA, that specifically bind to the molecule of interest. Finally, the position of the probe that is bound to the immobilized target molecule is visualized usually by autoradiography. -
Microchip Electrophoresis
Entry Microchip Electrophoresis Sammer-ul Hassan Mechanical Engineering, University of Southampton, Southampton SO17 1BJ, UK; [email protected] Definition: Microchip electrophoresis (MCE) is a miniaturized form of capillary electrophoresis. Electrophoresis is a common technique to separate macromolecules such as nucleic acids (DNA, RNA) and proteins. This technique has become a routine method for DNA size fragmenting and separating protein mixtures in most laboratories around the world. The application of higher voltages in MCE achieves faster and efficient electrophoretic separations. Keywords: electrophoresis; microchip electrophoresis; microfluidics; microfabrications 1. Introduction Electrophoresis is an analytical technique that has been applied to resolve complex mixtures containing DNA, proteins, and other chemical or biological species. Since its discovery in the 1930s by Arne [1], traditional slab gel electrophoresis (SGE) has been widely used until today. Meanwhile, new separation techniques based on electrophoresis continue to be developed in the 21st century, especially in life sciences. Capillary electrophoresis (CE) provides a higher resolution of the separated analytes and allows the automation of the operation. Thus, it has been widely used to characterize proteins and peptides [2], biopharmaceutical drugs [3], nucleic acids [4], and the genome [5]. The development of microfabrication techniques has led to the further miniaturization of electrophoresis known Citation: Hassan, S.-u. Microchip as microchip electrophoresis (MCE). MCE offers many advantages over conventional Electrophoresis. Encyclopedia 2021, 1, capillary electrophoresis techniques such as the integration of different separation functions 30–41. https://dx.doi.org/10.3390/ onto the chip, the consumption of small amounts of sample and reagents, faster analyses encyclopedia1010006 and efficient separations [6,7]. -
Fluorescent Proteins Filters, Mirrors and Wavelengths
White Paper Fluorescent Proteins Filters, Mirrors and Wavelengths By Bridget Bishop, Keri Raymond, Simone Rieger, and Paul Held, Applications Dept., BioTek Instruments, Inc. Fluorescent proteins have become a mainstay of today’s biomolecular research. Their small size, ease of use, wavelength variability and no substrate requirement make these genetic elements useful tools to answer countless numbers of experimental questions. Here we describe several of the commonly used technologies associated with fluorescent proteins. In addition an extensive list of fluorescent proteins, associated excitation and emission wavelengths and suggested filters and mirror combinations is provided. Introduction In the past 15 years, green fluorescent protein (GFP) has changed from a virtually unknown protein to a common molecular detection and imaging tool used in multiple scientific fields such as biology, chemistry, genetics, and medicine (Figure 1). The ability to auto-catalyze along with the relatively easy genetic encodability of GFP makes it ideal for minimizing the invasiveness of many procedures used to study biological processes. GFPs and GFP-like proteins (i.e., chromoproteins and other fluorescent proteins) are extremely useful due to their stability and also because their chromophore, (i.e. the protein region responsible for the color of GFP) is formed in an autocatalytic cycling of the 65SYG67 sequence (Figure 2). Because GFP doesn’t require a cofactor it can fluoresce under multiple conditions [1]. Figure 1. Green fluorescence in NIH3T3 cells expressing GFP. Originally discovered in the jellyfishAequorea victoria, GFP is a naturally fluorescent monomeric protein that is composed of 238 amino acids [2]. It is activated in A. Victoria by the naturally occurring bioluminescent protein aequorin, which releases blue light after binding with calcium. -
QPNC-PAGE Standardized Protocol for Acidic, Basic and Neutral Metalloproteins MW 6 - > 200Kda USE ONLY FRESH GELS
QPNC-PAGE Standardized Protocol For Acidic, Basic and Neutral Metalloproteins MW 6 - > 200kDa USE ONLY FRESH GELS Correspondence: Bernd Kastenholz • Research Centre Juelich • Institute of Chemistry and Dynamics of the Geosphere – Phytosphere (ICG-3) • Juelich 52425 • Germany • E-mail: [email protected] Recommended Tools (Bio-Rad) Model 491 Prep Cell (U.S. patent no. 4,877,510) Power Pac 1000: Constant Power: 5 W; Time: 8 hr Model EP-1 Econo Pump: 1 mL/min; 5 mL/fraction; 80 mL prerun V; 480 ml total V (Eluent) Model 2110 Fraction Collector: 80 Fractions Model EM-1 Econo UV Monitor: AUFS 1.0; Detection Wavelength 254 nm Model 1327 Econo Recorder: Range: 100 mV; Chart Speed: 6 cm/hr Model SV-3 Diverter Ventil Buffer Recirculation Pump: Flow Rate: 95 mL/min (Electrophoresis Buffer) Stock Solutions 1) 200 mM Tris-HCl 10 mM NaN3 pH 10.00 – Keep RT. 2) 200 mM Tris-HCl 10 mM NaN3 pH 8.00 – Keep RT. 3) 40 % Acrylamide/Bis 2.67 % C - Keep 4°C. 4) 10 % Ammonium Persulfate (APS) - Keep 4°C (freshly prepared). Electrophoresis Buffer 20 mM Tris-HCL 1 mM NaN3 pH 10.00 degassed – Keep 4°C Upper Electrophoresis Chamber (Prep Cell): 500 mL Electrophoresis Buffer Lower Electrophoresis Chamber (Prep Cell): 2000 mL Electrophoresis Buffer Eluent 20 mM Tris-HCL 1 mM NaN3 pH 8.00 degassed – Keep 4°C Elution Chamber (Prep Cell): 700 mL Elution Buffer Separating Gel Acrylamide 4% T Volume: 40 mL ingredients: 4 mL 40 % Acrylamide/Bis 2.67 % C 4 mL 200 mM Tris-HCl 10 mM NaN3 pH 10.00 32 mL H2O 200 µL 10% APS 20 µL TEMED Add TEMED and APS at the end. -
High Sensitivity Protein Gel Electrophoresis Label Compatible with Mass-Spectrometry
biosensors Brief Report High Sensitivity Protein Gel Electrophoresis Label Compatible with Mass-Spectrometry Joshua A. Welsh 1 , Lisa M. Jenkins 2 , Julia Kepley 1, Gaelyn C. Lyons 2, David M. Moore 1 , Tim Traynor 1, Jay A. Berzofsky 3 and Jennifer C. Jones 1,* 1 Laboratory of Pathology, Centre for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; [email protected] (J.A.W.); [email protected] (J.K.); [email protected] (D.M.M.); [email protected] (T.T.) 2 Laboratory of Cell Biology, Centre for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; [email protected] (L.M.J.); [email protected] (G.C.L.) 3 Vaccine Branch, Centre for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; [email protected] * Correspondence: [email protected] Received: 20 August 2020; Accepted: 28 October 2020; Published: 31 October 2020 Abstract: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely utilized technique for macromolecule and protein analysis. While multiple methods exist to visualize the separated protein bands on gels, one of most popular methods of staining the proteins is with Coomassie dye. A more recent approach is to use Bio-Rad stain-free technology for visualizing protein bands with UV light and achieve similar or greater sensitivity than that of Coomassie dye. Here, we developed a method to further amplify the sensitivity of stain-free gels using carboxyfluorescein succinimidyl ester (CFSE) staining. We compared our novel method using foetal bovine serum samples with Coomassie dye, standard stain-free gels, and silver staining. -
The Art of Protein Purification
1 The Art of Protein Purification William Ward Rutgers University, New Brunswick NJ, USA 1. Introduction Describing, in words, the details of protein purification to a relative novice in the field is not unlike explaining on paper the steps required to turn a set of colored oils into a beautiful pastoral scene on sheet of stretched canvas. Playing the oboe in a sophisticated metropolitan orchestra or performing a solo aria in a Gilbert & Sullivan operetta are accepted artistic endeavors that command great mastery of technique. Each of these art forms requires years of experience and endless experimentation and refinement of technique. Protein purification is no different. It is an art form. Like all other art forms, perfecting the art of protein purification requires a long apprenticeship. But, like all other art forms, protein purification is aesthetically rewarding to the practitioner. Every day brings new challenges, new insights, new hurdles, and new successes. Art is a process, not a destination. Protein purification fits the same definition. Perfecting the skills of protein purification can take many years of hands-on experience as well as periodic upgrading of those skills. Perhaps the most important part of protein purification is the set of pre-column steps that precede column chromatography. Pre- column steps are not covered as much in the protein purification literature as column chromatography, HPLC, and electrophoresis. So, I have chosen to focus much of my attention on the earlier stages of protein purification. More than column chromatography, pre-column steps are highly diverse and highly creative. Here the artistic aspects of protein purification are most apparent. -
Molecular Biologist's Guide to Proteomics
Molecular Biologist's Guide to Proteomics Paul R. Graves and Timothy A. J. Haystead Microbiol. Mol. Biol. Rev. 2002, 66(1):39. DOI: 10.1128/MMBR.66.1.39-63.2002. Downloaded from Updated information and services can be found at: http://mmbr.asm.org/content/66/1/39 These include: REFERENCES This article cites 172 articles, 34 of which can be accessed free http://mmbr.asm.org/ at: http://mmbr.asm.org/content/66/1/39#ref-list-1 CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» on November 20, 2014 by UNIV OF KENTUCKY Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Mar. 2002, p. 39–63 Vol. 66, No. 1 1092-2172/02/$04.00ϩ0 DOI: 10.1128/MMBR.66.1.39–63.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved. Molecular Biologist’s Guide to Proteomics Paul R. Graves1 and Timothy A. J. Haystead1,2* Department of Pharmacology and Cancer Biology, Duke University,1 and Serenex Inc.,2 Durham, North Carolina 27710 INTRODUCTION .........................................................................................................................................................40 Definitions..................................................................................................................................................................40 Downloaded from Proteomics Origins ...................................................................................................................................................40 -
Protein Blotting Guide
Electrophoresis and Blotting Protein Blotting Guide BEGIN Protein Blotting Guide Theory and Products Part 1 Theory and Products 5 Chapter 5 Detection and Imaging 29 Total Protein Detection 31 Transfer Buffer Formulations 58 5 Chapter 1 Overview of Protein Blotting Anionic Dyes 31 Towbin Buffer 58 Towbin Buffer with SDS 58 Transfer 6 Fluorescent Protein Stains 31 Stain-Free Technology 32 Bjerrum Schafer-Nielsen Buffer 58 Detection 6 Colloidal Gold 32 Bjerrum Schafer-Nielsen Buffer with SDS 58 CAPS Buffer 58 General Considerations and Workflow 6 Immunodetection 32 Dunn Carbonate Buffer 58 Immunodetection Workflow 33 0.7% Acetic Acid 58 Chapter 2 Methods and Instrumentation 9 Blocking 33 Protein Blotting Methods 10 Antibody Incubations 33 Detection Buffer Formulations 58 Electrophoretic Transfer 10 Washes 33 General Detection Buffers 58 Tank Blotting 10 Antibody Selection and Dilution 34 Total Protein Staining Buffers and Solutions 59 Semi-Dry Blotting 11 Primary Antibodies 34 Substrate Buffers and Solutions 60 Microfiltration (Dot Blotting) Species-Specific Secondary Antibodies 34 Stripping Buffer 60 Antibody-Specific Ligands 34 Blotting Systems and Power Supplies 12 Detection Methods 35 Tank Blotting Cells 12 Colorimetric Detection 36 Part 3 Troubleshooting 63 Mini Trans-Blot® Cell and Criterion™ Blotter 12 Premixed and Individual Colorimetric Substrates 38 Transfer 64 Trans-Blot® Cell 12 Immun-Blot® Assay Kits 38 Electrophoretic Transfer 64 Trans-Blot® Plus Cell 13 Immun-Blot Amplified AP Kit 38 Microfiltration 65 Semi-Dry Blotting Cells