Do you know how I can extract the detergent-insoluable protein from membrane proteins which will be used for co-immunoprecipitation assay? Thanks.
Helllo,
You can use Triton X100 for solubilization and afterwards apply ultracentrifugation. In supernatant you will find solubilized proteins and in pellet aggregated, insoluble proteins (see Bartoszewska et al, JBC, 2012).
greetings
The pellet that did not dissolve in the relatively mild detergent such as Triton X-100 may dissolve in SDS-PAGE sample buffer.
Good point. You could try other, less denaturing detergents to extract the post-Triton pellet, ones that would allow immunoprecipitation. Try zwitterionic detergents like Zwittergent 3-14 or CHAPS.
Otherwise you may try to solubilize in CHES or CAPS at high pH, either alone or, possibly better, with little amounts of sarcosyl. Let me know if it works.
You can also try to use a detergent like Dodeceyl D Maltoside (CMC of approx. 0.17mM). I tried it for the co-IP for 2 membrane proteins (TSPO and VDAC)
Another option is to solubilize the detergent-resistant material in 1-2% SDS and then dilute the SDS concentration to 0.1-0.2% before IP. Most antibodies tolerate 0.1-0.2% SDS. Whether your protein-protein association will survive solubilization in 1-2% SDS is a different question, however.
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