Best IPG strip rehydration method - (May/02/2009 )
I'd like to know the way you perform rehydration loading of samples into the IPG strip? Here is what I did, but seems not ideal:
first i add the sample buffer along the lower corner of a channel of the tray between the electrodes. The i place the strip (with the basic end first) to the liquid. Then I overlay them with 2ml mineral oil directly. All these steps are carried out on a flat surface.
i use the 17cm strip but i added 360 micro litre of sample buffer, so when i place the strip (i don't know whether it is the excess buffer or way i lower the strip incorrectly, the sample buffer overflow the electrodes and render even the non-gel edges of the strip filled with liquid.
i discover that there are still some minute bubbles trapped benneath the strip and cannot tap them away (especially near the + end)
Please kindly give me some advise. Thx in advance!
I do it in the same way, but i do it in the coffin. In coffin it becomes really easy, i use 250uL sample buffer for 13cm strip. And I overlay it with 2.5ml oil. sometimes I've observed that very minute bubbles at the end (only one or two) don't effect the gel, but these bubbles are hardly visible. what is the problem that you are facing in your gel? Has it not run properly?
amj2 on May 7 2009, 11:47 AM said:
Thx for ur reply. The problem i m facing is that during the lowering of the IPG strip onto channel of the tray, it is quite easy for the strip to touch the sample buffer (between the electrodes) and render a little bit of them spill out (still in the channel) but in the area outside the electrodes. I m worrying whether the proteins would be loaded into the strip as well.
Ok, I don't face this problem as i told you that I do it in coffin. But I have done it in manifold also. I hope you first place the electrodes and then put the sample buffer, try to put strip slowly. I know sometimes it happens that till the time you reach to another end of electrode, your sample buffer starts to spill. But this should not be too much, your protein will be definitely loaded on to strip. If you are not able to see it with coomassie stain, try to see it with silver stain.