Technical overviewIPG and 2D

Rehydrating the IPG strip

Loaded sample Applied IPG stripOverlay with mineral oil-prevents

Drying of the stripCrystallization of the ureaUptake of O2 and CO2

50 volts overnight

UVM Proteomics Outreach Module

Turned off IEF apparatus

Inserted pre-wet Wicks between electrode wire and IPG strip [both sides]

Wicks are required to absorb salts during the IEF

Turn on IEF apparatus on run the following cycle:250 volts 15 minutes8000 volts 2.5 hoursHold at 500 volts

Next Day…..

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Todays Lab: Running the 2D gel

Basically running a standard SDS-PAGE

Wash IPG strip in Equilibration buffer[s] (EB) containing:

Urea-chaotropic salt- disrupts non-covalent bonds includinghydrogen bondsVan der Waals Hydrophobic

SDS binds to protein and makes negatively charged

DTT Breaks disulfide bonds

Iodoacetimide -Alkylating sulfhydryl molecule

Binds to Cysteines Prevents Disulfide bridges

ProtocolRemove excess oil from IPG strip

Make sure EB1 and EB2 have their additives (DTT and Iodoacetamide)

Add EB1 buffer to strip [s] and rotate for 10 minutes

Transfer to EB2 and rotate for 10 minutes

Prepare the Bio-Rad PreCast Gel…..peel strips

Install IPG strip in the precast gel….+ end to the left!!!

Overlay the strip with melted agarose

Load standard and cover that with Agarose

Fill buffer chambers with Tris Glycine buffer

Run 45 mintes at 200Volts

Stain gel the same way as the previous SDS-PAGE [see page 23]