User:Jehane Ibrahim Eid/Notebook/Molecular Biology/2010/08/07

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  • Western blotting identifies with specific antibodies proteins that have been separated from one another according

to their size by gel electrophoresis. The blot is a membrane, almost always of nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed next to the membrane and application of an electrical current induces the proteins in the gel to move to the membrane where they adhere. The membrane is then a replica of the gel’s protein pattern, and is subsequently stained with an antibody. The following guide discusses the entire process of producing a Western blot: sample preparation, gel electrophoresis, transfer from gel to membrane, and immunostain of the blot. The guide is intended to be an educational resource to introduce the method rather than a benchtop protocol, but a more concise document suitable for consulting during an experiment can be composed by selecting and editing relevant material. Consult the manufacturers of your electophoresis and transfer equipment for more detailed instructions on these steps. A. Sample preparation 1. Lysis buffers 2. Protease and phosphatase inhibitors 3. Preparation of lysate from cell culture 4. Preparation of lysate from tissues 5. Determination of protein concentration 6. Preparation of samples for loading into gels B. Electrophoresis 1. Preparation of PAGE gels 2. Positive controls 3. Molecular weight markers 4. Loading samples and running the gel 5. Use of loading controls C. Transfer of proteins and staining (Western blotting) 1. Visualization of proteins in gels 2. Transfer 3. Visualization of proteins in membranes: Ponceau Red 4. Blocking the membrane 5. Incubation with the primary antibody 6. Incubation with the secondary antibody 7. Development methods

A. Sample preparation

1. Lysis buffers

To prepare samples for running on a gel, cells and tissues need to be lysed to release the proteins of interest. This solubilizes the proteins so they can migrate individually through a separating gel. There are many recipes for lysis buffers but a few will serve for most Western blotting experiments. In brief, they differ in their ability to solubilize proteins, with those containing sodium dodecyl sulfate and other ionic detergents considered to be the harshest and therefore most likely to give the highest yield. Most Abcam antibodies recognise reduced and denatured protein and should be used under reducing and denaturing conditions. It is important to note though that some antibodies will only recognize a protein in its native, non-denatured form and will not recognize a protein that has been extracted with a denaturing detergent (SDS, deoxycholate, and somewhat less denaturing, Triton X-100 and NP-40). The main consideration then when choosing a lysis buffer is whether the antibody one has chosen will recognize denatured samples. When this is not the case, it will be noted on the antibody datasheet, and buffers without detergent or with relatively mild non-ionic detergents (NP-40, Triton X-100) should be used. Protein Location And Lysis Buffer Choice Protein location Buffer recommended Whole Cell NP-40 or RIPA Cytoplasmic (soluble) Tris-HCl Cytoplasmic (cytoskeletal bound) Tris-Triton Membrane bound NP-40 or RIPA Nuclear RIPA or use nuclear fraction protocol* Mitochondria RIPA or use mitochondrial fraction protocol*

  • Proteins that are found exclusively or predominantly in a sub-cellular location can be enriched in a lysate of the

sub-cellular fraction compared to whole cell or tissue lysates. This can be useful when trying to obtain a signal for a weakly-expressed protein. For instance, a nuclear protein will be a larger proportion of the total protein in a nuclear lysate than it will be in a whole-cell or whole-tissue lysate, making it possible to load more of the protein per gel lane. Another advantage is the removal of potentially cross-reactive proteins present in the unused fractions. Please consult our separate protocols for sub-cellular fractionation.

Nonidet-P40 (NP40) buffer

150 mM sodium chloride

1.0% NP-40 (Triton X-100 can be substituted for NP-40)

50 mM Tris, pH 8.0

This is a popular buffer for studying proteins that are cytoplasmic, or membrane-bound, or for whole cell extracts. If there is concern that the protein of interest is not being completely extracted from insoluble material or aggregates, RIPA buffer may be more suitable, as it contains ionic detergents that may more readily bring the proteins into solution.

RIPA buffer (Radio Immuno Precipitation Assay buffer) 150 mM sodium chloride 1.0% NP-40 or Triton X-100 0.5% sodium deoxycholate 0.1% SDS (sodium dodecyl sulphate) 50 mM Tris, pH 8.0 RIPA buffer is also useful for whole cell extracts and membrane-bound proteins, and may be preferable to NP-40 or Triton X100-only buffers for extracting nuclear proteins. It will disrupt protein-protein interactions and may therefore be problematic for immunoprecipitations/pull down assays. The 10% sodium deoxycholate stock solution (5 g into 50 ml) must be protected from light. In cases where it is important to preserve protein-protein interactions or to minimize denaturation (for example, when it is known that the antibody to be used will only recognize a non-denatured epitope), a buffer without ionic detergents (e.g. SDS) and ideally without non-ionic detergents (e.g. Triton X-100) should be used. Cell lysis with detergent-free buffer is achieved by mechanical shearing, often with a Dounce homogenizer or by passing cells through a syringe tip. In these cases a simple Tris buffer will suffice, but as noted above, buffers with detergents are required to release membrane- or cytoskeleton- bound proteins.

Tris-HCl buffer 20 mM Tris-HCl, pH 7.5 Tris-Triton buffer: (Cytoskeletal proteins) 10 mM Tris, pH 7.4 100 mM NaCl 1 mM EDTA 1 mM EGTA 1% Triton X-100 10% glycerol 0.1% SDS 0.5% deoxycholate All four of these buffers will keep at 4oC for several weeks or for up to a year aliquotted and stored at -20oC. 2. Protease and phosphatase inhibitors As soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin. These events can be slowed down tremendously if samples are kept on ice or at 4°C at all times and appropriate inhibitors are added fresh to the lysis buffer. Ready-to-use cocktails of inhibitors from various suppliers are available but you can make your own cocktail. Inhibitor Protease/phosphatase Inhibited Final concentration in lysis buffer Stock (store at -20oC) Aprotinin Trypsin, Chymotrypsin, Plasmin 2 μg/ml D i l u t e in w a t e r, 10 mg/ml. Do not reuse thawed aliquots Leupeptin Lysosomal 5-10 μg/ml D i lu t e i n w a t e r . D o not re-use once defrosted. Pepstatin A Aspartic proteases 1 μg/ml D i l u t e in m e t h anol, 1mM. PMSF Serine, Cysteine proteases 1 mM Dilute in ethanol. You can re-use the same aliquot. EDTA Metalloproteases that require Mg++ and Mn++ 5 mM Dilute in dH2O, 0.5M. Adjust pH to 8.0. EGTA Metalloproteases that require Ca++ 1 mM Dilute in dH2O, 0.5M. Adjust pH to 8.0. Na Fluoride Serine/Threonine phosphatases 5-10 mM Dilute in water. Do not re-use once defrosted. Na Orthovanadate Tyrosine phosphatases 1 mM Dilute in water. Do not re-use once defrosted. Sodium orthovanadate preparation All steps to be performed in a fume hood. 1. Prepare a 100 mM solution in double distilled water. 2. Set pH to 9.0 with HCl. 3. Boil until colorless. Minimize volume change due to evaporation by covering loosely. 4. Cool to room temperature. 5. Set pH to 9.0 again. 6. Boil again until colorless. 7. Repeat this cycle until the solution remains at pH 9.0 after boiling and cooling. 8. Bring up to the initial volume with water. 9. Store in aliquots at - 20°C. Discard if samples turn yellow

3. Preparation of lysate from cell culture 1. Place the cell culture dish in ice and wash the cells with ice-cold PBS. 2. Drain the PBS, then add ice-cold lysis buffer (1 ml per 107 cells/100mm dish/150cm2 flask; 0.5ml per 5x106 cells/60mm dish/75cm2 flask). 3. Scrape adherent cells off the dish using a cold plastic cell scraper, then gently transfer the cell suspension into a pre-cooled microfuge tube. 4. Maintain constant agitation for 30 minutes at 4°C. 5. Centrifuge in a microcentrifuge at 4°C. You may have to vary the centrifugation force and time depending on the cell type; a guideline is 20 minutes at 12,000 rpm but this must be determined by the end-user (e.g. leukocytes need a very light centrifugation). 6. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice, and discard the pellet. 4. Preparation of lysate from tissues 1. Dissect the tissue of interest with clean tools, on ice preferably, and as quickly as possible to prevent degradation by proteases. 2. Place the tissue in round-bottom microfuge tubes or Eppendorf tubes and immerse in liquid nitrogen to “snap freeze”. Store samples at -80°C for later use or keep on ice for immediate homogenization. For a ~5 mg piece of tissue, add ~300 μl lysis buffer rapidly to the tube, homogenize with an electric homogenizer, rinse the blade twice with another 2x300 μl lysis buffer, then maintain constant agitation for 2 hours at 4°C (e.g place on an orbital shaker in the fridge). Volumes of lysis buffer must be determined in relation to the amount of tissue present (protein extract should not be too diluted to avoid loss of protein and large volumes of samples to be loaded onto gels. The minimum concentration is 0.1 mg/ml, optimal concentration is 1-5 mg/ml). 3. Centrifuge for 20 min at 12000 rpm at 4°C in a microcentrifuge. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice; discard the pellet. The buffer (with inhibitors) should be ice-cold prior to homogenization. 5. Determination of protein concentration Perform a Bradford assay, a Lowry assay or a BCA assay. Bovine serum albumin (BSA) is a frequently-used protein standard. Once you have determined the concentration of each sample, you can freeze them at -20°C or -80°C for later use or prepare for immunoprecipitation or for loading onto a gel.

Sample Buffer

  • Laemmli 2X buffer

4% SDS 10% 2-mercaptoehtanol 20% glycerol 0.004% bromophenol blue 0.125 M Tris HCl Check the pH and bring it to pH 6.8.

Sample Buffer

  • Laemmli 2X buffer

4% SDS 10% 2-mercaptoehtanol 20% glycerol 0.004% bromophenol blue 0.125 M Tris HCl Check the pH and bring it to pH 6.8.

6. Preparation of samples for loading into gels: denatured and native, reduced and non-reduced. a) Denatured, reduced samples Antibodies typically recognize a small portion of the protein of interest (referred to as the epitope) and this domain may reside within the 3D conformation of the protein. To enable access of the antibody to this portion it is necessary to unfold the protein, i.e. denature it. To denature, use a loading buffer with the anionic denaturing detergent sodium dodecyl sulfate (SDS), and boil the mixture at 95-100°C for 5 minutes. Heating at 70°C for 5-10 minutes is also acceptable and may be preferable when studying multi-pass membrane proteins. These tend to aggregate when boiled and the aggregates may not enter the gel efficiently. The standard loading buffer is called 2X Laemmli buffer, first described in Nature, 1970 Aug 15;227(5259):680-5. It can also be made at 4X and 6X strength to minimize dilution of the samples. The 2X is to be mixed in a 1:1 ratio with the sample.--Jehane Ibrahim Eid 03:22, 6 August 2010 (EDT)