suddenly IEF is not working - help!!!! (Jan/15/2008 )

Hi,

I am having a big problem in running IEF and am not able to figure out what the problem is, please anybody help me!!!
Actually when I run IEF, I give three steps in IPGPhor from amersham- step1-500v grad, step2-3500v grad, step3 3500v step, and 12 hours rehydration. I used to run IEF and it worked nicely but now when I am using same program in the same instrument , its not reaching to its optimum Vhs... don't know why . I thought the problem may be in strip, but I tried the working strips from other labs.. dint work, Then I thought problem may be in rehydration buffer, made fresh buffer .. again it did not work. I've tried all possible things but its not working. rehydration buffer has urea thiourea and CHAPS. Has anyone ever faced the same problem?? please help me in this... I don't know what to do??????

Thanks

-mysterious-

have you tried cleaning the electrodes? are they making proper contact?

does the power supply reach the set voltage? at the same speed?

you rehydrate after the run? not before?

i'm confused. the way i do it, the ipg strips come dry, you rehydrate then apply sample and run.

if the run is not reaching the proper volthours then continue the run until it does. this doesn't correct the underlaying problem but it will allow you to get usable results.

-mdfenko-

I also do the same thing, I rehydrate first for 12 hours and then set the program. To check the power supply and all, I ran only rehydration buffer in the coffin... I added dry strip cover fluid in this and set the voltage 1000V for 1 hour. I did this with the old and new buffer both. With the old buffer it could reach upto 250V only but with the new buffer it could reach to 1000V, and 500VhS. Then I thought the problem was with rehydration buffer. So again I lysed the cells, did acetone precipitation and resuspended the pellet in new rehydration buffer. but in the morning when I came....... its not working. what do I do???????? I feel like some ghost has come into the lab..... who just don't want to see me happy .

-mysterious-