1. Do you keep the ice box in the hood or outside while washing with PBS and adding lysis buffer?
2. How much time you leave lysis buffer before scraping?
3. How much time you centrifuge?
i searched high and low on Internet and papers but everyone used something different.
Hi,
I saw that you have many questions regarding the PAGE. Go through this manual to find out answers to most of your questions.
http://www.abcam.com/ps/pdf/protocols/wb-beginner.pdf
For WB troubleshooting you can refer the following
http://web.mnstate.edu/provost/ProteinBlottingBook.pdf
Probably the reason for so much variation in methods is that the result isn't very sensitive to the details of the method. I suggest you try a few different conditions for each parameter you think will matter (long versus short incubation, long versus short centrifugation) and see what works best for you. I doubt it matters whether the ice box is inside or outside the hood.
Keeping the samples in icebox outside shouldn't be a problem. No need of hood. If you homogenize using pestle, one hour in lysis buffer with protease inhibitors, in ice should be fine. If the yield is still less, then u can try sonication. Centrifugation: 12000 rpm for 15-20 minutes at 4 degree should be sufficient.
This is the most thorough, stepwise and updated western blot guidelines. It should have all the information you need: http://www.currentprotocols.com/WileyCDA/CPUnit/refId-im0810.html
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