i tried to extract the urine protein from human urine sample by applying the methodology of TCA-Acetone precipitation (20% TCA in acetone). The precipitate obtained after overnight -20C incubation was centrifuged @ 7000g for 20 mins @ 4C. The pellet obtained was washed with 20mM DTT in chilled Acetone (-20C) and washed again with -20C chilled acetone without DTT this time.

Then I allowed the acetone to evaporate and resuspended the pellet in milliQ water. Then I filtered it through 3KDa molecular weight cut off (MWCO) Amicon filters from millipore using milliQ water thrice and finally collected the concentrated sample (approx. 80ul) by buffer exchanging it with rehydration buffer (8M urea, 2% CHAPS, 30mM tris-Cl- pH8.5). Now I got the concentration of proteins approx. 4.5ug/ul. Now the problem is that when I introduced this proteins to IEF focussing using a 7 cm IPGstrip, it does not show a perfect run in the program. There is a gradient run in my IEF program of 5000V for 15000Vh; in this gradient run the samples has not achieved even 2000V total and it run for the rest of the run at below 1500V. I am not understanding where is the problem. As far as i know I have done enough to desalt my sample using the MWCO filters, then why my sample didn't take the complete 5000V gradient focusing? If anyone has a working IEF program for 7cm IPG Strips please take the pain to send it to me.