I want to isolate total proteins from mouse brain tissues to run western blots & determine levels of nuclear-rich proteins such as p16INK4a and other cytoplasmic proteins. I used RIPA (50mM Tris (pH 7.4), 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10mM NaF, 1mM EDTA) supplemented with protease & phosphatase inhibitor to homogenize, followed by sonication, rest on ice for 30 min, followed by centrifugation at various g force & duration. According to my Western blot results, there are bands of p16INK4a but only in the pellets, and not the supernatant, when I centrifuge either at low speed (1000g x 10min) or high speed (16000g x 20 min). How can I keep p16 proteins in the supernatant?
RIPA can have poor efficiency with specific sample types, see this useful review https://www.lubio.ch/assets/PDFs/Invent_RIPA_Buffer-_The_Potential_Troubles.pdf
Sample prep solution for 2D-PAGE experiments called 2D rehydration buffer which includes non-ionic detergents and urea may work. Recipe like;
(8 M urea, 4% CHAPS, 40 mMDTT)...You may also modify RIPA by increasing the SDS, adding a reducing agent, and adding urea as chaotropic agent up to 8M to induce solubilization...You may also investigate novel methods for increased solubilization at inclusion bodies...
here is one of them;
https://www.thermofisher.com/order/catalog/product/78115?gclid=Cj0KCQjwrfymBhCTARIsADXTabmpmgARsfk_UEnrzRvAvjpRGkKZ2y3Er3Wnqn4PTAXuMAaDtbzLxiYaAuQmEALw_wcB&ef_id=Cj0KCQjwrfymBhCTARIsADXTabmpmgARsfk_UEnrzRvAvjpRGkKZ2y3Er3Wnqn4PTAXuMAaDtbzLxiYaAuQmEALw_wcB:G:s&s_kwcid=AL!3652!3!458037844736!!!g!!!6617619314!79549275115&cid=bid_pca_ppf_r01_co_cp1359_pjt0000_bid00000_0se_gaw_dy_pur_con
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