SDS-polyacrylamide gel electrophoresis

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SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique in biochemistry and molecular biology for separating proteins based on their molecular weight. This method allows scientists to analyze the composition of complex protein mixtures and determine the relative molecular mass of unknown proteins.

Purpose

(1) To understand the principle behind SDS-polyacrylamide gel electrophoresis.

(2) To master the technique of determining the relative molecular mass of proteins using this method.

Principle

Sodium dodecyl sulfate (SDS) is an anionic detergent that binds to proteins, denaturing them and giving them a uniform negative charge. This process masks the natural charge differences between proteins, allowing separation based solely on size. As a result, the mobility of proteins during electrophoresis depends primarily on their molecular weight, enabling accurate determination of unknown proteins through comparison with standard protein markers.

Materials and Equipment

Reagents:

  • 30% Separation Gel Stock Solution: 30g Acr, 0.8g Bis in 100mL non-ionized water
  • 10% Concentrated Gel Stock Solution: 10g Acr, 0.5g Bis in 100mL non-ionized water
  • Separation Gel Buffer: 48mL HCl, 36.3g Tris in 100mL ion-free water
  • Concentrated Gel Buffer: 48mL HCl, 5.98g Tris in 100mL ion-free water
  • Electrophoresis Buffer: 1.6g Tris, 28.8g Glycine, 1g SDS in 1L ion-free water
  • Sample Loading Buffer: 100mg SDS, 0.1mL thiol ethanol, 1mL glycerol, 2mg bromophenol blue, 0.5mL 0.2mol/L phosphate buffer, diluted to 10mL with distilled water
  • Coomassie Brilliant Blue Staining Solution: 0.25g R-250, 454mL 50% methanol, 46mL glacial acetic acid
  • Decolorization Solution: 75mL glacial acetic acid, 875mL water, 50mL methanol
  • 10% Ammonium Persulfate, 10% SDS, 1% TEMED

Equipment:

  • DYCZ-24D Vertical Electrophoresis Tank
  • Pipettes: 1mL (×3), 5mL (×4), 0.5mL (×1), 0.1mL (×2)
  • Beakers: 100mL (×2)
  • Thin Pipette Tips, Microsyringes

Procedure

  1. Assemble the Electrophoresis Tank: Place a silicone sealing frame on a flat glass plate, then position the concave glass plate over it. Insert the comb into the sample well and check for leaks with distilled water.
  2. Prepare the Separation Gel: Mix 5mL of 30% gel stock, 2.5mL of pH 8.9 Tris-HCl buffer, 0.2% SDS, and 10.2mL deionized water. Add 1% TEMED and 10% ammonium persulfate, mix well, and pour into the gel chamber. Cover with distilled water to ensure even polymerization.
  3. Prepare the Concentrated Gel: Mix 3mL of 10% gel stock, 1.25mL of pH 6.7 Tris-HCl buffer, 1% TEMED, and 4.6mL deionized water. Pour into the upper part of the gel chamber and allow it to solidify.
  4. Sample Preparation: Dissolve each protein in sample buffer at 0.5mg/mL, heat in boiling water for 3 minutes, and cool before loading.
  5. Loading: Load 10–15μL of sample into the wells using a microsyringe. Ensure all wells are filled before starting the run.
  6. Electrophoresis: Apply 10mA initially, increasing to 20–30mA once samples enter the separation gel. Stop when the bromophenol blue dye reaches the bottom.
  7. Staining and Destaining: Incubate the gel in Coomassie stain for 1 hour, rinse with water, and destain until bands are clear.
  8. Data Analysis: Measure migration distances and plot log(molecular weight) vs. relative mobility to determine the molecular weight of unknown proteins.

Precautions

  1. Each run should use its own standard curve for accurate results.
  2. Soak the gel in decolorizing solution before staining to remove SDS and reduce background staining.
  3. If the sample is aqueous, double the concentration before mixing with the sample buffer.

This detailed procedure ensures precise and reproducible results, making SDS-PAGE a cornerstone technique in protein analysis.

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