I have expressed a membrane protein in Pichia pastoris and can purify it with DDM or TritonX-100. However when I use 2%CHAPS(30-40mM,with CMC 8mM) to dissolve membrane fragment I failed. But I have to purify it by CHAPS in my next step. Anyone has good ideas?
Hi Laqiwe, TX-100 is a nonionic polyethylene glycol-based detergent, DDM is a nonionic glycosylated fatty acid-based detergent.
CHAPS is a zwitterionic cholesterol-based detergent.
Can you use nonionic cholesterol-based detergents such as:
Digitoxigenin?
http://www.sigmaaldrich.com/catalog/product/sigma/d9404?lang=en®ion=US
or Digitonin?
http://www.sigmaaldrich.com/catalog/product/sigma/d5628?lang=en®ion=US
If you cant use those detergents, you could try sonicating your sample with the CHAPS to force your protein into those micelles.
Sometimes 4% CHAPS works better (65mM) or maybe a combination like used here:
http://www.proteomesci.com/content/pdf/1477-5956-3-5.pdf
(In some cultures the finger display in your photo might be seen as offensive)
thats better with the cropping, good luck!
Can you keep the protein, initially in the triton, then do ion exchange, bind your protein to the column, wash and exchange the detergent for CHAPS before continuing?
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