Western Blot SDS-PAGE

LOADING AND RUNNING THE GEL

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.

With our automated Simple Western™ instruments, all steps following sample preparation and plate loading are automated. Simple Western uses capillary electrophoresis to separate your samples and then your samples are covalently immobilized to the capillary wall. The steps of antibody binding and detection are then precisely controlled inside an automated benchtop instrument, providing fully quantitative and reproducible results with flexible multiplex detection strategies and total protein normalization. Learn more about our Simple Western instruments from ProteinSimple, a Bio-Techne brand.

SDS-PAGE Gel Electrophoresis Protocol

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



Protein Size	Gel Percentage
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.



2. 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.


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



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



4. Run the gel as recommended by the manufacturer.
   
   Note: 1-2 hours at 100 V is standard, but time and voltage may require optimization.