ProteomicsProteomicsDay 3 Tech TalkDay 3 Tech Talk

ActivitiesActivities1.1. Prepare IPG strip for second gel Prepare IPG strip for second gel electrophoresiselectrophoresis

2.2. Run IPG strip in second dimension SDS-Run IPG strip in second dimension SDS-PAGE electrophoresis gel. PAGE electrophoresis gel.

3. Stain with Coomassie Blue3. Stain with Coomassie Blue

Equilibration of IPG stripEquilibration of IPG strip

Equilibration buffer (EB): 6M urea and 30% Equilibration buffer (EB): 6M urea and 30% glycerol--aids transfer of proteins from IPG glycerol--aids transfer of proteins from IPG strip to second dimension gel.strip to second dimension gel.

immerse in EB with DTT—preserves the fully immerse in EB with DTT—preserves the fully reduced state of denatured, unalkylated reduced state of denatured, unalkylated proteins.proteins.

immerse in EB with iodoacetamide— alkylates immerse in EB with iodoacetamide— alkylates thiol groups on proteins, preventing their thiol groups on proteins, preventing their reoxidation during electrophoresis.reoxidation during electrophoresis.

Setting up precast gelSetting up precast gel

Remove gel cassette Remove gel cassette from its packagefrom its package

It has one long slot for It has one long slot for the IPG strip and one the IPG strip and one short well for the protein short well for the protein standardsstandards

Peel off protective strip Peel off protective strip on bottom of gel and on bottom of gel and remove green well remove green well comb.comb.

Place in electrophoresis Place in electrophoresis chamberchamber

Placing the IPG strip in the gel slotPlacing the IPG strip in the gel slot

Using forecepts, place the IPG strip Using forecepts, place the IPG strip between the plates on the surface of between the plates on the surface of the second-dimension gel with “+” the second-dimension gel with “+” end on the left and the label end on the left and the label “readable”.“readable”.

Gently push the IPG strip down so Gently push the IPG strip down so the entire lower edge of the IPG strip the entire lower edge of the IPG strip is in contact with the top surface of is in contact with the top surface of the slab gel .the slab gel .

Ensure that no air bubbles are Ensure that no air bubbles are trapped between the IPG strip and trapped between the IPG strip and the slab gel surface or between the the slab gel surface or between the gel backing and the glass plate.gel backing and the glass plate.

Agarose overlay of IPG stripAgarose overlay of IPG strip

Imbedding the IPG strip in agarose prevents it from Imbedding the IPG strip in agarose prevents it from moving or floating in the electrophoresis buffer. And moving or floating in the electrophoresis buffer. And connects it to the gel.connects it to the gel.

Heat the agarose (which contains bromo-phenol blue Heat the agarose (which contains bromo-phenol blue dye) in the microwave oven until agarose melts. Let it dye) in the microwave oven until agarose melts. Let it cool down for 5 minutes before using. cool down for 5 minutes before using.

Slowly pipette the amount required to seal the IPG strip Slowly pipette the amount required to seal the IPG strip in place. Pipet slowly to avoid introducing bubbles. Allow in place. Pipet slowly to avoid introducing bubbles. Allow a minimum of 1 minute for the agarose to cool and a minimum of 1 minute for the agarose to cool and solidify. solidify.

Don’t cover protein standard well.Don’t cover protein standard well.

Ready to runReady to run

Add protein standard to its well. Overlay Add protein standard to its well. Overlay with agarose.with agarose.

Add buffer to the chamber as indicated.Add buffer to the chamber as indicated.

Run electrophoresis at 200 volts for 45 Run electrophoresis at 200 volts for 45 minutes, until dye front is at bottom of gel. minutes, until dye front is at bottom of gel. It is OK if dye front elutes off bottom.It is OK if dye front elutes off bottom.

Remove gel from cassette and stain with Remove gel from cassette and stain with Coomassie blue as before.Coomassie blue as before.