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Problem with protein extraction from rat hippocampus

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#1 FJB



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Posted 30 May 2011 - 01:38 PM

Hi ppl! hope you can help me with this....

Im trying to extract total proteins from hippocampal tissue from rat days: embryonic 18, postnatal 10, postnatal 30, and postnatal 90. I use a RIPA buffer consisting in 50mM Tris, 150mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS, + protease inhibitors.

To quantify the proteins I have used both Bradford (Biorad; not recommended because of the detergents in the buffer) and BCA (Pierce) making 1/10 and 1/5 dilutions. After doing the standard curve with BSA (0-8 ug) my proteins show very similar concentrations in the range of 4-5 ug/ul. Ive taken 20ug or 30ug starting from this concentrations to run a gel/WB. The problem is that when I incubate with loading control antibodies such as GAPDH, B-tubulin, alpha-tubulin, alpha-actinin, or n-cadherin (yes, I have tried then all!!), I get huge variations on the resulting bands!!! Im talking about 10 times difference between samples!!

Any suggestions on how to fix this?? different extraction buffers?? Has anyone tried sonicating fresh samples??


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