Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a fundamental technique used to separate proteins based on their molecular weight. This method is essential for protein analysis, providing information about protein size, purity, and relative abundance in complex biological samples.

Here we go over the SDS-PAGE protocols, including the reagents, solutions, procedures, and troubleshooting advice for typical issues. Our aim is to help you understand and explain the protocol processes and take full advantage of the detailed protocols for western blot.

Solutions and Reagents

Workflow

Workflow of SDS-PAGE

Troubleshooting

Despite the simplicity of the SDS-PAGE, problems may still arise such as poor band resolution. For some of the problems that may occur, we provide the following troubleshooting guide.

• Poor Band Resolution

Common causes include incorrect acrylamide concentration for target protein size - select appropriate percentage based on molecular weight range; running the gel at too high voltage causing heat generation - reduce voltage or use cooling system; buffer depletion during long runs - replace with fresh buffer periodically; old or improperly stored acrylamide solutions affecting polymerization - use fresh reagents stored properly; inadequate sample denaturation - ensure complete heating and SDS binding.

• Smeared or Distorted Bands

Sample overloading can overwhelm gel capacity - reduce protein amount loaded; presence of nucleic acids or lipids in samples - perform additional purification steps; incomplete protein solubilization - optimize sample buffer composition or add solubilizing agents; high salt concentration interferes with migration - desalt or dilute samples; trapped air bubbles affect protein migration - degas solutions and pour carefully.

• Vertical Streaking

Protein precipitation in sample wells often results from insufficient SDS or high salt content - adjust buffer composition accordingly; inadequate sample heating causes incomplete denaturation - ensure proper heating time and temperature; protein aggregation may occur - consider adding urea or other solubilizing agents; contamination of wells with polyacrylamide fragments - clean wells thoroughly before loading; improper loading technique causes spillover - use proper loading tips and technique.

• Uneven Migration Across Gel

Uneven polymerization results from improper mixing of gel solution - mix thoroughly before pouring; temperature gradients during electrophoresis - ensure consistent temperature or use cooling; uneven buffer levels between chambers - check and adjust buffer volumes; electrical field distortion from buffer leaks - check apparatus for leaks; warped gel plates create uneven thickness - use properly leveled casting stand.

• No Bands Visible

Insufficient protein loading requires concentration adjustment - increase sample volume or concentrate proteins; proteins not retained during staining - check staining protocol and timing; expired or improperly prepared staining reagents - use fresh solutions; protein degradation from protease activity - add protease inhibitors during sample preparation; proteins running off gel - select appropriate gel percentage for target molecular weight.

• Ghost Bands

Carry-over contamination from previous samples requires thorough cleaning between runs; degradation products indicate proteolysis - add protease inhibitors and work quickly; non-specific staining of gel impurities - filter gel solutions before casting; artifacts from improper gel handling - use clean equipment and handle carefully; contaminated reagents or buffers - prepare fresh solutions under clean conditions.

• Wavy Band Patterns

Trapped air bubbles during gel casting create uneven patterns - degas solutions and pour carefully; uneven polymerization from temperature variations - ensure consistent room temperature; electrical field disturbances from buffer discontinuities - check buffer composition and pH; mechanical vibration during run - place apparatus on stable surface; improper comb insertion causes gel distortion - insert comb straight and level.

• Poor Staining

Insufficient staining time or incorrect concentration requires protocol optimization; SDS interference with dye binding - include destaining step to remove excess SDS; over-destaining removes protein-bound dye - monitor destaining process carefully; incompatibility between protein type and staining method - try alternative staining protocols; expired or improperly prepared staining solutions - use fresh reagents stored properly. Insufficient staining time or incorrect concentration requires protocol optimization; SDS interference with dye binding - include destaining step to remove excess SDS; over-destaining removes protein-bound dye - monitor destaining process carefully; incompatibility between protein type and staining method - try alternative staining protocols; expired or improperly prepared staining solutions - use fresh reagents stored properly.

Products with Tested Data

At Creative Biolabs, we are dedicated to providing high-quality antibodies for various research applications. Each product in our extensive range has been rigorously tested to ensure superior reliability and efficacy. To showcase the performance of our antibodies, we have conducted numerous experiments using SDS-PAGE. Below, you will find a table listing a selection of our antibody products along with images from these SDS-PAGE experiments, demonstrating their proven reliability.

For research use only. Not intended for any clinical use.