Problem with 2D

[prot]

Hi. I am doing my proteomics work on fungus. Do anyone have any good references for this kind of work? Journal and note on protein extraction, 2d run, troubleshooting tip...I have been running my samples currently using 7cm IPG strip from biorad. I have some problem with it. First, my IEF run took unusual long time- more than 10hr to complete. Is there anything I can do to improve it? What is the running voltage should we use? And I noticed the proteins are being focused in white line on the strip. Is this normal? I normally store my strip before the 2nd dimension. Do we need to wash it and cover with mineral oil for storage? I prepared my own agarose for sealing the srip. I found that the agarose sometimes solidify before I place my strip nicely. And bubbles trap easily between the strip and gel. Any suggestion to correct this? Besides, I use silver stain to visualize the protein spots. Sometimes, I found some clear spots that are not stained with the silver stain. What is the problem actually? And my final 2d image normally appear with streaking. Please help me.