After sample preparation, samples in loading buffer must be loaded into a gel. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) basically separates proteins according to their molecular weight (MW), based on their rates of migration through the polyacrylamide gel under the influence of an applied electrical field. SDS is a detergent present in the SDS-PAGE sample buffer where, along with a bit of heat and a reducing agent, it makes the protein linear and uniformly negative charged, and ensures it stays that way all throughout the procedure. After the separation of proteins by SDS-PAGE, proteins will be transferred from the gel to the nitrocellulose membrane or PVDF. Refer to Protein Transfer from Gel to Membrane in Western Blot Assay for blotting steps.
SDS-PAGE 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.
Common 1X Running Buffer Recipes
Adjust pH to 8.3
Acrylamide gels can be prepared at different concentrations. As a general rule, low MW proteins are best resolved on high percentage gels, whereas larger proteins require lower percentage gels for sufficient resolution.
|Protein Size||Gel Percentage|
|4-40 kDa||Up to 20%|
Note: If the MWs of your targets range from low to high MW sizes or if you plan to probe a blot for multiple proteins varying in size, gradient gels may be necessary to achieve efficient separation of proteins.
Note: Voltage may vary depending on research needs. 1 hour at 100V is a standard guideline.
Important Tips -What to add to your run other than the samples themselves?
Positive control is something you need to incorporate into your experiments-especially when using new protocols or antibodies. This way you will be certain that your antibody is actually capable of binding the protein of interest, even though it might not be present in the analyzed samples.
Loading controls are a way of confirming that protein loading is equal across the gel, and their expression level should not vary between the different samples. Housekeeping genes are often used as loading controls because those are the proteins that exhibit high-level, constitutive expression in the cell type or sample we are analyzing. It is important to select a loading control that has a different MW than the protein of interest-otherwise, you won’t be able to distinguish the bands.
More WB Resource