Can any one provide me appropriate protocol for preparing crude mouse tissue lysate for western blotting. The protein of interest resides both in cytoplasm and nucleus so which lysis buffer would be appropriate?
- I routinely used to make lysates of rat and mouse tissue for Western blotting
- As mentioned RIPA buffer with protein inhibitors is perfectly good for this application
- Furthermore as mentioned I would suspend your tissue in ICE COLD inhibitors and RIPA buffer and homogenise
- I attach my old SOP
RIPA buffer with protease inhibitors (and phosphatase inhibitors, if you are looking at phospho proteins). You probably need to sonnicate the lysate as well.
I agree with Mirsada, RIPA buffer solubilizes all membranes. However, RIPA buffer breaks interactions between proteins, so it also depends what the purpose of your experiment is.
If you don't need nuclear proteins and if you want to preserve protein-protein interactions and protein structure integrity I would recommend either NP40 or TritonX buffer. CHAPS buffer is a bit stronger than NP40 and TritonX, but preserves some protein interactions.
I resuspend the tissue in the buffer, keep the sample on ice for 30' and resuspend every 10'. You can also use a homogenizer/sonicator. Spin down @ 13200rpm, and collect the supernatant.
- I routinely used to make lysates of rat and mouse tissue for Western blotting
- As mentioned RIPA buffer with protein inhibitors is perfectly good for this application
- Furthermore as mentioned I would suspend your tissue in ICE COLD inhibitors and RIPA buffer and homogenise
- I attach my old SOP
Dear Dr. Laurence Stuart Dawkins-Hall ,
I saw that you made approx. 20% homogenate from rat tissue for western blot as you have mentioned in the protocol (used ~5ml RIPA for 1g of tissue). Do you have reference in support of it? It will be very helpful if you provide it here.
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