Proteomics Day 3 Tech Talk. Activities 1.Prepare IPG strip for second gel electrophoresis 2.Run IPG strip in second dimension SDS- PAGE electrophoresis.

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Proteomics Day 3 Tech Talk

Activities 1.Prepare IPG strip for second gel electrophoresis 2.Run IPG strip in second dimension SDS- PAGE electrophoresis gel. 3. Stain with Coomassie Blue

Equilibration of IPG strip Equilibration buffer (EB): 6M urea and 30% glycerol--aids transfer of proteins from IPG strip to second dimension gel. immerse in EB with DTT—preserves the fully reduced state of denatured, unalkylated proteins. immerse in EB with iodoacetamide— alkylates thiol groups on proteins, preventing their reoxidation during electrophoresis.

Setting up precast gel Remove gel cassette from its package It has one long slot for the IPG strip and one short well for the protein standards Peel off protective strip on bottom of gel and remove green well comb. Place in electrophoresis chamber

Placing the IPG strip in the gel slot Using forecepts, place the IPG strip between the plates on the surface of the second-dimension gel with “+” end on the left and the label “readable”. Gently push the IPG strip down so the entire lower edge of the IPG strip is in contact with the top surface of the slab gel. Ensure that no air bubbles are trapped between the IPG strip and the slab gel surface or between the gel backing and the glass plate.

Agarose overlay of IPG strip Imbedding the IPG strip in agarose prevents it from moving or floating in the electrophoresis buffer. And connects it to the gel. Heat the agarose (which contains bromo-phenol blue dye) in the microwave oven until agarose melts. Let it cool down for 5 minutes before using. Heat the agarose (which contains bromo-phenol blue dye) in the microwave oven until agarose melts. Let it cool down for 5 minutes before using. Slowly pipette the amount required to seal the IPG strip in place. Pipet slowly to avoid introducing bubbles. Allow a minimum of 1 minute for the agarose to cool and solidify. Don’t cover protein standard well.

Ready to run Add protein standard to its well. Overlay with agarose. Add buffer to the chamber as indicated. Run electrophoresis at 200 volts for 45 minutes, until dye front is at bottom of gel. It is OK if dye front elutes off bottom. Remove gel from cassette and stain with Coomassie blue as before.