IEF-PAGE stands for Iso-electric focusing polyacrylamide gel electrophoresis.
Here is the equipment you will need.
This is the Pharmacia PhastSystem that you will be using to run the electrophoresis.
First, cover the area with saran wrap.
Turn on the instrument.
Clean the gel compartment with a small amount of dd water.
Also clean the plastic cover and insert into place on the electrode assembly for IEF-PAGE.
For IEF-PAGE, the eccentric lever should be at the low position such that
the electrode can lay flat on the gel surface.
Press the SEP stand by temp button and press DO to cool the gel bed.
Cut open the packaging of the PhastGel IEF.
Both sides of the gel are protected by plastic covering.
The top plastic cover is much thinner than the bottom plastic.
While the thinner top plastic cover is to be removed prior to running the gel, the thicker bottom plastic should stay intact.
Check that the thin plastic cover is on top.
Then using a tweezer, bend the plastic tab of the gel upwards to identify the side that is facing up.
Apply a small amount of dd water onto the separation bed.
Gently lay the gel onto the separation bed, thicker plastic side down, starting from the top and slowly down the bottom.
Try your best to avoid forming air bubbles under the gel.
Remove as much excess dd water as you can by touching the edge of the gel with kimwipes.
You should do this until the gel cannot be easily moved
and will stay within the position outlined by the red box.
With a tweezer, lift the thin plastic protective film on the gel.
The gel has an anode end and a cathode end. The cathode end of the gel is indicated by the protruding plastic tab.
Lower the electrode assembly onto the gel surface and finally the sample applicator arm.
Close the cover.
To prepare the samples, put a piece of parafilm on the sample-well stamp
and lightly press against it being careful not to puncture the parafilm.
This will prevent the wells from leaking when the samples are loaded.
Press until the wells show up on the parafilm.
Lift the protective cover on the parafilm and you can load samples onto the wells.
Quickly vortex the fish extracts then pipette 5 microlitres of sample onto the well.
The drop should form a nice dome shape without air bubbles.
Once all the samples have been placed,
remove the 8/1 sample applicator from the Zip-loc bag.
8/1 refers to 8 lanes, each with 1 microlitre.
Align the 8 teeth of the applicator with the 8 wells on the parafilm
and gently touch the sample to obtain 1 microlitre.
Open the cover of the PhastSystem, and gently place the sample applicator in the middle slot of the sample applicator arm and close the cover.
By placing the comb in the middle of the gel,
proteins with an isoelectric point greater than 7 will migrate towards the cathode
while proteins with an isoelectric point below 7 will migrate towards the anode.
Press SEP-start, 1 or 2 representing 1 or 2 gels, and then DO.
Then press 3 representing IEF 3 - 9 then DO to start the run.
Once the run is complete, the machine will make a sound to alert you.
Press SEP-start, then DO to end the run.
Lift the sample applicator arm and electrode assembly so you can remove the gel.
Place the gel in a square petri dish, add the fixing solution
and leave for 5 minutes at room temperature.
Discard the fixing solution into the acid waste
then add the washing solution and leave for 2 minutes at room temperature.
Discard the washing solution, add Coomassie blue staining solution,
place in a metal tray and incubate at 50°C for 15 minutes.
Continue to stain until you can observe distinct bands on the gels.
Discard the staining solution, add the destaining solution, and incubate at 50°C for 10 minutes.
Include a folded sheet of KimWipe or absorbent paper towel in the petri-dish to help absorb excess dye.
When the gel has completed destaining to a transparent background with distinct blue bands,
scan the gel and analyze using the densitometer.
The gel can be kept by replacing the plastic cover on the gel,
then stored in a small Zip-loc bag.
Carefully clean the PhastSystem with dd water and kimwipes, then unplug.
The staining and destaining solution should be properly disposed of into the non-halogenated waste container.