Western Blot

WESTERN BLOTTING - A BEGINNER’S GUIDE
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 tomove 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 methodrather 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 fromcell 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 gels2. 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

D. References 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 sothey 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 antibodiesrecognise 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, www.abcam.com/technical

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 andNP-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 Whole Cell Cytoplasmic(soluble) Cytoplasmic (cytoskeletal bound) Membrane bound Nuclear Mitochondria Buffer recommended
NP-40 or RIPA

Tris-HCl Tris-Triton NP-40 or RIPA RIPA or use nuclear fraction protocol* 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 ortissue 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...