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SDS Page Protocol

SDS-PAGE Protocol for Protein Analysis and Antibody Validation

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a widely used technique for separating and analyzing proteins based on their molecular weight (MW) and a fundamental tool in the field of molecular biology and protein research. This protocol outlines the steps to prepare the samples and run a gel SDS-PAGE.

Materials:

  • Protein samples and molecular weight (MW) markers to estimate the molecular weight of your samples.
  • Running Buffer: Tris-Glycine-SDS buffer is more suitable for a broader range of protein sizes, while Tris-Tricine-SDS buffer offers improved resolution for smaller proteins. Both running buffers ensure protein denaturation, charge uniformity, and efficient protein separation during electrophoresis.
  • Acrylamide Gels: Prepare polyacrylamide gels with the desired percentage for your specific experiment
    • Note: The choice of gel percentage depends on the MW of the target protein. Lower percentage gels like 4-20% separate a wide range of protein sizes, with smaller proteins migrating faster and larger proteins slower. Higher percentage gels, such as 15%, are employed for resolving smaller proteins with greater precision. Intermediate percentages like 10% and 12% are versatile choices for routine protein separations.
  • Loading Buffer: Mix your protein samples with SDS-PAGE loading buffer. Different recipes and variations of sample loading buffers may exist for specific research purposes, but Laemmli buffer is a widely used and versatile option for routine SDS-PAGE experiments. Typical loading buffers include a reducing agent (e.g., DTT or β-mercaptoethanol) to break the disulfide bonds and denaturing proteins, which is essential for accurate molecular weight determination; SDS to impart a uniform negative charge to the proteins and a tracking dye to provide a visual reference during electrophoresis. 
  • Electrophoresis apparatus and appropriate power supply.

After running the gel, the protein bands can be visualized with Coomassie Blue or Silver for gel staining. Alternatively, the gel can be transferred to nitrocellulose or PVDF membranes for subsequent Western Blotting.

*Make sure to use personal protective equipment, such as gloves and lab coats.

Procedure:

1. Prepare the acrylamide gel according to your specific requirements (percentage and thickness). Assemble the gel in the gel cassette, insert the comb, and allow it to polymerize.

2. Mix the protein samples and molecular weight markers with loading buffer. Heat the samples at 95 °C for 5 minutes to denature proteins. Load the samples into the wells of the gel alongside molecular weight markers.

3. Place the gel cassette into the electrophoresis chamber. Fill the chamber with running buffer. Apply a constant voltage (typically 100-200 V) and run the gel until proteins have separated according to their molecular weights (this can vary depending on gel type and target proteins).

4a. After electrophoresis, carefully remove the gel from the cassette. Stain the gel with Coomassie Blue or another appropriate stain for visualization. Examine the gel to observe protein bands and compare the migration of your samples to the molecular weight markers.

OR

4b. Transfer the gel to a membrane for Western Blot analysis.

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