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Technical overview IPG and 2D

Technical overview IPG and 2D. UVM Proteomics Outreach Module. Rehydrating the IPG strip Loaded sample Applied IPG strip Overlay with mineral oil-prevents Drying of the strip Crystallization of the urea Uptake of O2 and CO2 50 volts overnight. Next Day….

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Technical overview IPG and 2D

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  1. Technical overviewIPG and 2D

  2. UVM Proteomics Outreach Module Rehydrating the IPG strip Loaded sample Applied IPG strip Overlay with mineral oil-prevents Drying of the strip Crystallization of the urea Uptake of O2 and CO2 50 volts overnight

  3. Next Day….. 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 minutes 8000 volts 2.5 hours Hold at 500 volts -20

  4. 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 including hydrogen bonds Van 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

  5. Protocol Remove 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]

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