ProteomicsProteomicsDay 2 Tech TalkDay 2 Tech Talk

ActivitiesActivities

1.1. Run 1 dimension SDS-PAGE gel of Run 1 dimension SDS-PAGE gel of proteins isolated on day one and stain with proteins isolated on day one and stain with Coomassie BlueCoomassie Blue

2.2. Rehydrate protein pellet and start first Rehydrate protein pellet and start first dimension separation using iso-electric dimension separation using iso-electric focusing (IEF). We will use an IPG focusing (IEF). We will use an IPG ((iimmobilized mmobilized ppH H ggradient) strip for this radient) strip for this separation. separation.

Part 1: 1D SDS-PAGE gelPart 1: 1D SDS-PAGE gel

Run protein on 1D SDS-PAGE gelRun protein on 1D SDS-PAGE gelSample is labeled 1D—contains 12 ul Sample is labeled 1D—contains 12 ul

Add 3 ul of protein loading buffer to the sampleAdd 3 ul of protein loading buffer to the sample

Place samples in a beaker of boiling water or 98C Place samples in a beaker of boiling water or 98C thermocycler for 5 minutesthermocycler for 5 minutes

Load 10 ul of the sample into gel well.Load 10 ul of the sample into gel well.Run 10 minutes @ 50 volts—gets proteins lined up at Run 10 minutes @ 50 volts—gets proteins lined up at separating gel interfaceseparating gel interface

Run 35 minutes @ 150 volts—separates proteins by sizeRun 35 minutes @ 150 volts—separates proteins by size

Stain with Coomassie blue for 45 minutesStain with Coomassie blue for 45 minutes

Destain with dd water twice (10 minutes). Let sit overnight in Destain with dd water twice (10 minutes). Let sit overnight in dd waterdd water

Part 2 IEFPart 2 IEF

Sample is labeled “2D” and contains Sample is labeled “2D” and contains protein pellet from 200 ul of extracted protein pellet from 200 ul of extracted protein mixtureprotein mixture

Use rehydration buffer to dissolve pellet. Use rehydration buffer to dissolve pellet.

Need to add 300 ug of protein to IPG stripNeed to add 300 ug of protein to IPG strip

Rehydration– use formula to determine Rehydration– use formula to determine volume of rehydration buffer to use.volume of rehydration buffer to use.

Formula for rehydrating protein Formula for rehydrating protein pelletpellet

Want 300 ug of protein on IPG strip—will add 200ul of 1.5 Want 300 ug of protein on IPG strip—will add 200ul of 1.5 ug/ul sampleug/ul sample

Your protein pellet was made from 200ul of protein extract.Your protein pellet was made from 200ul of protein extract.

Formula: multiply 200 by your measured protein Formula: multiply 200 by your measured protein concentration and divide this value by 1.5. This will give concentration and divide this value by 1.5. This will give you the ul volume of rehydration buffer to use.you the ul volume of rehydration buffer to use.

Example:Example: Your measured protein concentration is 1.8 Your measured protein concentration is 1.8 ug/ul.ug/ul.

1.8 x 200 = 360 ug protein. 360 / 1.5 = 240 ul 1.8 x 200 = 360 ug protein. 360 / 1.5 = 240 ul

Use 240 ul of rehydration buffer to rehydrate your protein Use 240 ul of rehydration buffer to rehydrate your protein pellet.pellet.

Set up the IPG stripSet up the IPG stripTransfer 200ul of your rehydrated protein sample to Transfer 200ul of your rehydrated protein sample to channel of IEF tray. Keep track of which channel your channel of IEF tray. Keep track of which channel your sample is insample is in

Add IGP strip to the tray with + signs lined up. Record Add IGP strip to the tray with + signs lined up. Record the number of the IPG strip in your notebook.the number of the IPG strip in your notebook.

Cover the strip with mineral oil.Cover the strip with mineral oil.

Run the “rehydration” program over night.Run the “rehydration” program over night.

IEF—to be done by course instructor the next day.IEF—to be done by course instructor the next day.

Wet electrode wicks in waterWet electrode wicks in water

Insert the wicks between the electrode wire and IPG Insert the wicks between the electrode wire and IPG strip on both sidesstrip on both sides

Run IEF, wrap tray in plastic wrap and store tray in Run IEF, wrap tray in plastic wrap and store tray in freezerfreezer