Gel Electrophoresis of Proteins
Samples to be run on native gels should be prepared in a way that minimizes the denaturation of the proteins. Avoid heat, strong detergents, foaming, and over-dilution. In addition, the activity of endogenous proteases must be kept to a minimum. Keeping the sample cold and including protease inhibitors will be helpful in this regard. A formulation for a cocktail of protease inhibitors with a broad spectrum of activity is given below:
Preparation of Protease Inhibitor Cocktail (100X)
Mix the following:
- 200µg/ml aprotinin and antipain
- 100µg/ml pepstatin A
- 25µg/ml leupeptin and chymostatin
- 15mM benzamidine
Store at -70°C for up to 2 months.
Cell and Tissue Disruption
Tissue culture cells and soft tissue such as the liver can be prepared by homogenizing 10 – 15 strokes in a Dounce homogenizer. The homogenizer produces micro-turbulent regions which tear cells apart but are not sufficient to disrupt a strong collagen matrix. Some grinding also occurs, which allows soft tissues to be prepared.
The choice of the buffer is dictated by the requirements of the protein of interest, although some general principles apply. Isotonic (100 – 150mM salt) buffers of pH 6.5 – 8.5 are best for most applications. Large deviations from this range may destabilize proteins, and will also introduce artifacts into the electrophoresis results. Tris or phosphate buffers work well in this pH range.
Bacteria and more structurally strong tissues can be disrupted by sonication. Ultrasonic waves produce rapidly alternating high and low-pressure waves, disrupting cells by shear force and cavitation. This generates high local heat output, so the sonication must be carried out on the ice, in short bursts separated by relatively long recovery times. Another important consideration with this procedure is that positioning the probe too close to the solution surface may cause foaming, which can extensively denature sample proteins.
For tough materials, the French Press is appropriate. This device forces the material to be disrupted through a narrow opening at extremely high pressure. This generates shear forces and pressure differentials which tear apart most biological samples. The cell can be kept cold and flow rates controlled to minimize foaming, so this is a good method for the recovery of active proteins.
Other methods for disrupting cells to recover active proteins include enzymatic digestion, grinding in liquid N2, grinding in alumina, and disruption in a blender. The topic is reviewed well in “Protein Purification” by Robert K. Scopes.
NEXT TOPIC: Immuno-Electrophoresis / Immuno-Diffusion
- Sample Preparation for SDS-PAGE
- Sample Preparation for Native Protein Electrophoresis
- Peptide Mapping
- Native Protein Electrophoresis
- Measuring Molecular Weight with SDS-PAGE
- Isoelectric Focusing
- Immuno-Electrophoresis / Immuno-Diffusion
- Gel Preparation for Native Protein Electrophoresis
- Gel Electrophoresis of RNA & Post Electrophoretic Analysis
- Denaturing Protein Electrophoresis: SDS-PAGE
- Casting Gradient Gels
- Activity Stains
- Sample Preparation
- Electrophoresis Buffers
- Blotting and Detection Buffers
- Gel Matrices
- Gel Accessories
- Ultra Pure Reagents