I'm using 4% stacking and varying from 7.5% to 11% separating gels (Tris-HCl). I'm getting decent resolution but the dye front is extremely wonky.. I don't really understand why. My first reaction was that it was a leak, but i put the unit back together multiple times the first time it happened and it continued to occur. I've had this problem numerous times now, I've checked for leaks every single time from the inner chamber but they definitely don't exist. So anyone have any ideas? Major thanks, I'm feeling pretty stupid about this.


Edit: Oh, and I've tried a variety of different voltages, I usually just use 100V though.

Couldn't seem to attach image, so link:
http://users.tpg.com.au/adslixvi/gel-from-1-05-and-3-05-munt-crop.jpg

Try to download the image once again please.

There could be couple of reasons to this peoblem.
First, the polimerization of the gel is not 100%. Try to buy precated gel to see if the same problem acures.
Second, Problem with the voltage, staking usualy at 60v and seperating 120v. Look at the Amperage to see if it is not stable there is a problem with the aparatus.
Try to see if the runnig buffer is getting warmer then it should be, if yes try to change it.
Third, sometimes when the protein that is loaded in the welles are at different total protein and/or different total velumes this could be the resone.

So first try to load same amount of volume and same amount of total pretein.
Then try the other two point I mentioned.

Best of Luck
Guy

Thanks, I think I'll just go ahead with precast gels.

Remember that the pre cast are a bit expencive and they last for 3 month (shelf life), store them at 2-8deg in a horizontal position.
Please let me know if it solved your problem.
Guy

Hi, yeah the precast gels are working fine, I get worse resolution though. I think some of the APS I was using was the problem, I'm going to try making all completely fresh solutions/buffers and seeing what happens. Thanks again

Great to hear the good news,
The problem with the precast gels is a common one. It is always important to remember the the APS should be fresh can stay at 4deg up to 6 weeks.
One way to know that the APS is not good is to take time for the polymerization if it is more the 30 min for the running portion the APS is Bad.
Guy[/quote]

I freeze my APS solution in aliquotes at -20°C and I use them even when they are 1 year old and I never have problems. To me it seems safer then to keep the APS solution at 4°C.
Gibacht

If it works it works.
I always prepare fresh APS which last for one month.
Guy

can anyone has protocol for pegylation of insulin?

I think that this should have its own thred as it relates to change in a surface / biomaterials.
In any case try to see the article from this link:
http://www.liebertonline.com/doi/abs/10.1089/107632704323061799?cookieSet=1&journalCode=ten

Guy