I would be very grateful if anyone had any input on what the problems are with my 2D gel. Gel A is human synovectomy tissue and Gel B is a cell line.The synovectomy sample was previously stored in RNAlater so I dialysed and did an acetone precipitation then rehydrated in standard 2d buffer. There is 200ug protein loaded on to an 11cm pH4-7 Bio rad strip. The IEF protocol is as follows:
1-step 500V 1hr.
2-gradient 1000V 1 hour.
3-gradient 6000V 2.5 hours
4- step 6000V 50 minutes.
Gel A is the worst.There are excessive amounts of vertical streaking. Also, there is a line across the gels at the bottom, even though the dye front was run right to the bottom of the gels .
Has anyone any ideas what could be causing the streaking and the line across the bottom?