Proteomics
Day 2 Tech Talk
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.
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
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.
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.
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