I am interested in studying the interaction of outer mitochondrial membrane bound proteins by co-immunoprecipitation. Which would be the most suitable detergent for this study: Digitonin Vs CHAPS Vs Triton?
I have used Triton X-100 (1% and below) or Tween-20 (1% and below) for co-IP experiments with good success. I've not used CHAPS or digitonin so I can't really compare how they will affect results relative to triton. The use of a non-ionic detergent vs a zwitter ionic detergent depends on your particular system. In my experience, saponin-based detergents are generally used for permeability of membranes that will still leave the overall membrane structure intact (for IF staining, etc) or as a mild additive to minimize non-specific binding in immunoassays.
I recommend starting with a lysis and wash buffer that contains 1% TX-100, and adjusting the concentration or testing other detergents if you see contamination. It all depends on particular proteins you are trying to detect via co-IP.
You may also consider adding glycerol to your extraction buffer to improve protein stability.
http://sevierlab.vet.cornell.edu/resources/CalbiochemDetergentsBook.pdf
Another option could be isolating mitochondria before doing IP.
https://tools.thermofisher.com/content/sfs/manuals/MAN0011504_Mitochondria_Isolat_CulturedCells_UG.pdf
Hi,
Its a good option to start first with isolating mitochondria. Also certain membrane protein donot solubilize in non denaturing condition such as in 1% TX-100. You need to use some chaotropic agents along with detergent to solubilize all the protein irrespective whether it is transmembrane or membrane. But this will break all protein-protein interaction (PPI).
IP only with TX-100 mostly works out for Strong PPI and ones that are soluble in TX-100. You can check whether you protein is soluble in TX-100 or not.
This the protocol I used to study trasmembrane protein interaction in the nuclear envelope, some of the resistant proteins such as Intermediate filaments can be solubilized, but I crosslink all the proteins just before lysis.
1. Isolate mitochondria or take whole cell pellet
2. Add 7M UREA and 1% TX-100 (say 100-200ul) and incubate 20 mins on ice, or 5 times the size of the pellet, use Protease inhibitor in all the step. -- Lysate
3. Since such high concentration will degrade you antibody, if you directly do IP. Dilute the above mixture 7 or 8 times, so the concetration of TX-100 is less than 0.2% and urea is less than 1M. So your final volume of lysate after diluting will be 1.5 to 2.0 ml. With this volume you can do IP.
Check this paper
http://www.ncbi.nlm.nih.gov/pubmed/24950247
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