Gel Electrophoresis of Proteins
Sample Preparation for SDS-PAGE
SDS is a powerful detergent, which will solubilize many cells and tissues. This greatly facilitates sample preparation for SDS PAGE because most samples will be completely dissolved by heating to 95°C in loading buffer (detailed below). A somewhat stronger loading buffer, containing more SDS and Dithiothreitol (DTT) at a higher pH, can be used for the homogenization of more difficult samples.
In general, the goal of sample preparation is to denature the proteins fully, to disrupt any disulfide bonds through reduction, and to dissolve any particles which would interfere with electrophoresis. Incomplete denaturation will not fully saturate the proteins with SDS and will lead to blurred bands or altered mobilities. Failure to dissolve sample particulate completely will result in clogging of the gel which will cause streaking from the well to the end of the gel. Disulfide reduction is often required to release subunits from multimeric proteins. In many instances, it is instructive to run samples with and without reduction, to demonstrate which bands are released by disulfide disruption.
Sample Preparation for SDS-PAGE Electrophoresis
Prepare 2X sample buffer consisting of 0.5M Tris-HCl, pH 6.8, 4.4% SDS, 300mM Mercaptoethanol, 10mg/ml Bromophenol Blue and mix with an equal volume of sample (For greater reproducibility, employ National Diagnostics preformulated 2X sample buffer, Protein Loading Buffer Blue 2X). Bring to 95° C for 10 minutes, and cool to room temperature before loading. If particulate is present, centrifuge samples for 5 minutes at 14k RPM in a microcentrifuge, and load the gel.
The previous protocol is sufficient for most tissue culture cells, fluid samples (such as serum and cerebrospinal fluid), bacteria, and some soft tissues. For more highly structured samples, use the following buffer:
Homogenize samples in 5 – 15 volume of CHES buffer (consisting of a mixture of 1% CHES pH to 9.5 with NaOH, 2% SDS, 1% DTT and 10% glycerol that has been stored at -20° C up to 6 months.) in a Dounce homogenizer at room temperature, 25 – 50 strokes.
Heat homogenate to 95°C for 5 minutes and allow to cool. (homogenized samples will contain substantial amounts of cellular debris, which must be removed by centrifugation to avoid clogged wells. Centrifuge samples @ 14K RPM in a microcentrifuge for 15 minutes.
NOTES ON YEAST AND BACTERIA: these organisms may be encapsulated in a layer of lipopolysaccharide (LPS) which will require enzymatic digestion with Lysozyme or Zymolyase prior to homogenization.
NEXT TOPIC: Gel Preparation for SDS-PAGE
- 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