1. Proteomics Day 2 Tech Talk

2. Activities 1.Run 1 dimension SDS-PAGE gel of proteins isolated on day one and stain with Coomassie Blue 2.Rehydrate protein pellet and start first dimension separation using iso-electric focusing (IEF). We will use an IPG (immobilized pH gradient) strip for this separation.

3. Part 1: 1D SDS-PAGE gel Run protein on 1D SDS-PAGE gel Sample is labeled 1D—contains 12 ul Add 3 ul of protein loading buffer to the sample Place samples in a beaker of boiling water or 98C thermocycler for 5 minutes Load 10 ul of the sample into gel well. Run 10 minutes @ 50 volts—gets proteins lined up at separating gel interface Run 35 minutes @ 150 volts—separates proteins by size Stain with Coomassie blue for 45 minutes Destain with dd water twice (10 minutes). Let sit overnight in dd water

4. Part 2 IEF Sample is labeled “2D” and contains protein pellet from 200 ul of extracted protein mixture Use rehydration buffer to dissolve pellet. Need to add 300 ug of protein to IPG strip Rehydration– use formula to determine volume of rehydration buffer to use.

5. Formula for rehydrating protein pellet Want 300 ug of protein on IPG strip—will add 200ul of 1.5 ug/ul sample Your protein pellet was made from 200ul of protein extract. Formula: multiply 200 by your measured protein concentration and divide this value by 1.5. This will give you the ul volume of rehydration buffer to use. Example:Your measured protein concentration is 1.8 ug/ul. 1.8 x 200 = 360 ug protein. 360 / 1.5 = 240 ul Use 240 ul of rehydration buffer to rehydrate your protein pellet.

6. Set up the IPG strip Transfer 200ul of your rehydrated protein sample to channel of IEF tray. Keep track of which channel your sample is in Add IGP strip to the tray with + signs lined up. Record the number of the IPG strip in your notebook. Cover the strip with mineral oil. Run the “rehydration” program over night. IEF—to be done by course instructor the next day. Wet electrode wicks in water Insert the wicks between the electrode wire and IPG strip on both sides Run IEF, wrap tray in plastic wrap and store tray in freezer