IEF gels - samples stuck in wells

[STEFFF]

I have a problem with IEF, my samples do not leave the wells, maybe they are precipitated, I don't know. So, here is was I am doing. I hope, that anybody has an idea what is going wrong here..

I cast my own IEF gels:

2.7 mL water
2.4 mL 50 % glycerol
2 mL Acrylamide/ Bisacrylamide 37:1
0.6 mL Ampholytes 4-6
6 g Urea
40 µL Triton-X
25 µL 10 % APS
20 µL TEMED

The protein dilution buffer is 8M Urea/ 4 % CHAPS/ 0.5 % ampholytes, 0.5 % Triton-X, 20 mM DTT.

I dilute the protein in buffer about 15-30 minutes before I apply it on the gel. Then, I mix it with glycerol (15 %) and apply it on the gel. I fill the wells of the gel with dilution buffer + 15 % glycerol as overlay.

Then I fill the upper chamber with 25 mM phosphoric acid and the outer chamber with 50 mM NaOH. The chamber is connected to the power supply so that the polarity is reversed. Then I run the gel for 1h at 150 V, follwed by 1 h at 200 V and over night at 250 V (this is the maximum I can reach with our power supply).

Oh, and I pre-run the gel for 30 minutes at 150 V with dilution buffer in the wells (w/o glycerol). After the pre-run, I empty the chamber completely, rinse the wells with water and apply the sample.

So, can anybody help me?

I never did IEF before and puzzled this protocol out. But obviously, it is not very good...