although I saw spesific binding before. now I don't see any spesific bind. everything that I use is same but I didn't see at the latest a few assays. What could be the problem? Is it perishable homogenates? my homogenates have been waiting for about 6 months and without proteases and B-mercaptoethanol
Western blot non-specific bands?
although I saw spesific binding before. now I don't see any spesific bind. everything that I use is same but I didn't see at the latest a few assays. What could be the problem? Is it perishable homogenates? my homogenates have been waiting for about 6 months and without proteases and B-mercaptoethanol
Western blot non-specific bands?
I have basic one question? how I save primary and secondary antibodies which I used it before.
Antibodies binding quite also depend on their LOT. the batch of their production. If you are using the same batch as you used before, then maybe you have problems of protein degradation. Maybe you can try with fresh homogenates, with protease inhibitors added. Some proteins are not very stable in liquid suspension.
Please see some information about WB problems resolution:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/
https://medschool.vanderbilt.edu/cdb-core-lab/files/cdb-core-lab/public_files/083013_WESTERN%20BLOTTING%20TIPS%20AND%20TROUBLESHOOTING%20GUIDE.pdf
http://www.cellsignal.com/common/content/content.jsp?id=western-trouble
http://www.abcam.com/protocols/western-blot-troubleshooting-tips
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