We tried to follow a published protocol where authors claim they can co-immunoprecipitate protein complexes from lipid rafts with RIPA buffer (1% NP-40, 0.1% SDS) but we cannot reproduce their results. Immunoprecipitations work well but protein complexes are probably disrupted. We would like to switch to milder detergents but we don't really know if they would be efficient in solubilizing membrane rafts.
I work on gamma-secretase which is an enzymatic complex made of four subunits that are enriched in lipid rafts. 1% CHAPSO buffer is commonly used to co-immunoprecipitate gamma-secretase subunits as it preserves the integrity of the complex. Considering the efficiency of different detergents for solubilizing lipid rafts protein, I recommend the attached article from my former colleague Vetrivel. I hope it could help you.
Here is the link to the article if the attachment does not work: http://www.ncbi.nlm.nih.gov/pubmed/15322084
Membrane protein IP assay is a little difficult. I have found many protocol about it. at last, I use one as follow. You can use SDS-buffer (100-200ul) to lyse the cell, and then add 800 ul NP-40 buffer to dilute it. here you can IP you protein easily and effectively.
I usually use PS1 NT antibody for gamma secretase IP (rabbit antiserum raised against residues 1-65 of PS1, described in Thinakaran et al., Neurobiology of Disease, 1998: http://www.ncbi.nlm.nih.gov/pubmed/9666482)
Is there anyone with evidences in different results obtained by using Nonidet-P40 or Brij98 in sample buffer to detect proteins localized in detergent resistant membranes by western blotting?
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