Western blot SDS-PAGE

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Western blot handbook

Troubleshooting guide

Recommended loading controls

Sample preparation

Running the SDS-PAGE gel

Transferring protein from gel to membrane

Immunoblotting and detection

Membrane stripping and reprobing

Blot storage

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After sample preparation, samples in loading buffer must be loaded onto a gel. Proteins in the sample are separated from each other based on their size by SDS-PAGE gel electrophoresis. Electrophoresis is performed with a negative pole (cathode) on one end of the gel and a positive pole (anode) on the opposite end of the gel. The negatively charged SDS bound to proteins causes migration of protein complexes towards the positive pole (anode) during electrophoresis, allowing proteins to be separated by size. In general, the larger the protein, the slower it migrates through the gel. Acrylamide gels can be prepared at different concentrations. As a general rule, low molecular weight proteins are best resolved on high percentage gels, whereas large proteins require lower percentage gels for sufficient resolution.

SDS-PAGE Protocol

  1. Prepare or purchase a pre-made gel of appropriate polyacrylamide percentage to best resolve your protein of interest based on molecular weight.

    4-40 kDa Up to 20%
    12-45 kDa 15%
    10-70 kDa 12.5%
    15-100 kDa 10%
    50-200 kDa 8%
    >200 kDa 4-6%

    Note: If your protein of interest has multiple isoforms ranging from low to high molecular weight sizes or if you plan to probe a blot for multiple proteins varying in size, gradient gels (acrylamide concentration increases from top to bottom) may be necessary to achieve efficient separation of proteins.

  3. Load samples containing equal amounts of protein (10-50 µg protein from cell lysate or 10-100ng purified protein) prepared in sample buffer into SDS-PAGE wells. Include a molecular weight marker in one of the lanes.

  4. Fill the electrophoresis apparatus with 1X running buffer as instructed by the manufacturer.

  5. Buffer Components
    1X Running buffer 25 mM Tris base
    192 mM glycine
    0.1 % SDS
    Adjust pH to 8.3

  6. Run the gel as recommended by the manufacturer. Voltage may vary depending on research needs. 1 hour at 100V is a standard guideline.

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