Hi to everybody. I am planning to perform a pulldown assay, but I am not sure about what detergent should I use in my lysis buffer. I want to pull down an endosomal membrane protein with a cytosolic protein.
What are the main differences between both detergents in terms of disrupting endosomal membranes?
Thanks a lot!
Both detergents can solubilise membranes.
Triton is a non-ionic detergent, which bears no electrical charge. It is relatively mild, that means, it will preserve both specific and unspecific protein-protein interactions. In other words, your chances of detecting the interaction you want are quite high, but the result will not be very clean.
CHAPS is a zwitterionic detergent, that has both a positively charged quaternary amino-group and a negatively charged sulphonic acid group. Its power to interrupt protein-protein interactions is higher than that of Triton. Your gels will come out cleaner, but the chances to miss specific effects is higher (you recall the relationship between errors of first and second kind?).
One possibility is to solubilise first with Triton only, and then optimise the assay by adding CHAPS (and/or SDS) until your blot comes out clean, but without too much loss of your signal.
For such experiments a "sparse matrix" (Taguchi) design can save you a lot of time.
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