Procedure for 2D SDS gel electrophoresis
Two-dimensional gel electrophoresis is a powerful analytical tool for complex protein samples such as those from cellular lysates. The technique relies on one isoelectric focusing step, which separates protein on the basis of isoelectric point, followed by a second separation based on denaturing SDS gel electrophoresis, which resolves proteins on the basis of molecular weight.
- For the 1st dimension, IEF strip gels pH 3-10NL (Pharmacia Biotech) are used.
- IEF Strips are rehydrated in 120ul of sample in the 2D sample buffer ( 8M urea, 2% CHAPS, 2% IPG Buffer pH3-10NL, 0.3%DTT and trace amounts of Bromophenol Blue).
- Iso Electric Focusing is done on the Pharmacia IEF Apparatus. The gel is focused overnight at a given voltage (3000V).
- The gel strip is equilibrated for the 2nd dimension in solution 1 and 2 for 10min each. Prepare Equilibration Stock Solution (10% Tris-Cl solution pH6.8., 36% Urea, 30% Glycerol, 1%SDS). Add 25-100mg DTT/10mlequilibration solution for the 1st equilibration step and 0.25g iodoacetamide plus a few grains of Bromophenol Blue /10ml solution for the 2nd step.
- The gel strip is loaded on the SDS NuPAGE 2D gel and electrophoresis done at a fixed voltage using appropriate buffer for definite length of time.
- The gel is then removed from cassette and fixed, stained and destained using Coomassie blue or Silver stain.
- The desired protein band is cut from the gel using a sharp razor blade. Further cut this band into 1mm pieces.
- The protein is then digested using trypsin In Gel digestion procedure.
Contributed by Bob Lee
, Ph.D., Director of Protein Laboratory, Research Resources Center at UIC.