can anyone help me what brain homogenate concentration would be better if i want to detect Total CREB, pCREB, Total BAD and pBAD through WB from brain tissue of hamsters infected with Prion 263K?
Hello,
What do you mean with 10 % ? I use brain homogenate with a concentration around 3 mg/ml minimum to get sure you can load your SDS page with max 15 to 20 ul (homogenate + Laemmli buffer). The loading average volume is about 30 ug of protein to be detected in WB
no you got me wrong...i am not talking about final concentration in well...i simply mean which concentration of homogenate is usually used when we homogenise brain tissue? i mean brain to RIPA buffer ratio....1;10?
Syed, If you are asking how dilute to make your lysate a 1:10 ratio of tissue weight (mg) to lysis buffer (uL) is a reasonable starting point to end up with a total protein concentration that will fit nicely into your gel wells. In rat brain I usually use 1:5 dilution to get about 5-10 ug/uL total protein concentration (measured with a BCA assay). Using a quick spin of the tissue to remove excess water, I can use a 1:10 ratio. It will take some experimentation, as your lysis buffer recipe and homogenization method will affect how well the tissue will lyse. Many protocols suggest 1:20 ratio, but in my experience with rat midbrain, that ends up much too dilute requiring too much lysate volume for loading 20-50ug of protein per lane. I haven't worked with CREB or BAD but my efforts with other phosphor-protein antibodies required a moderately large amount of whole cell lysate to get a signal there (e.g. 30-50ug). Best of luck.
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