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 resol es proteins on the basis of molecular weight. !. "or the !st dimension, #$" strip gels p% &-!'() *Pharmacia +iotech, are used. 2. #$" Strips are rehydrated in !2'ul of sample in the 2D sample buffer * -. urea, 2/ 0%1PS, 2/ #P2 +uffer p%&-!'(), '.&/DTT and trace amounts of +romophenol +lue,. &. #so $lectric "ocusing is done on the Pharmacia #$" 1pparatus. The gel is focused o ernight at a gi en oltage *&'''3,. 4. The gel strip is equilibrated for the 2nd dimension in solution ! and 2 for !'min each. Prepare $quilibration Stoc5 Solution *!'/ Tris-0l solution p%6.-., &6/ 7rea, &'/ 2lycerol, !/SDS,. 1dd 28-!''mg DTT9!'mlequilibration solution for the !st equilibration step and '.28g iodoacetamide plus a few grains of +romophenol +lue 9!'ml solution for the 2nd step. 8. The gel strip is loaded on the SDS (uP12$ 2D gel and electrophoresis done at a fixed oltage using appropriate buffer for definite length of time. 6. The gel is then remo ed from cassette and fixed, stained and destained using 0oomassie blue or Sil er stain. :. The desired protein band is cut from the gel using a sharp ra;or blade. "urther cut this band into !mm pieces. -. The protein is then digested using trypsin #n 2el digestion procedure. 0ontributed by +ob )ee , Ph.D., Director of Protein )aboratory, <esearch <esources 0enter at 7#0.