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prot 12-31-2009 10:18 AM

2d problem
 
Hi, everyone. I am new in the proteomics field. I am currently running 2d gel for my fungus sample. I have some problem with it and hope that anyone can advice me.
1) Firstly, my IEF take unusual long time to focus the 7cm strip- more than 10hr. Is this acceptable or how to improve it? My friend run his E.coli sample using the same IEF unit but didn't encounter the same problem. Maybe problem with my sample preparation?
2) And I normally store my strip before 2nd dimension run. Do we need to wash and cover the strip with mineral oil for storage?
3) I am using my own agarose to seal the strip. But I found that my agarose sometimes solidify before I can place my strip properly. And this traps small bubbles in between also. How to avoid this?
4) Finally I stained my 2d gel using silver stain. I notice that sometimes there are some clear spot that are unstained. What should I do?

I appreciate if anyone can comment and advice me. Thanks a lot.


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