I tagged a GPCR with an HA-Tag.
Now I want to do Co-IP. all the protocols I found are designed for soluble proteins. I was wondering if anyone has experience in this field. I know every membrane receptor needs different solubilization conditions but maybe there is already a good protocol !

Thanks

This should work let me know how it turns out.

Rb

http://www.proteinscience.org/cgi/content/full/14/10/2638

"Solubilization was carried out at 4°C. Frozen cells (80 g) were homogenized in 600 mL of solubilization buffer (50 mM Tris-HCl at pH 7.5, 200 mM NaCl, 0.5% [w/v] CHAPS, 0.1% [w/v] CHS, 1% [w/v] DM, 5 mM MgCl2, and 30% glycerol) supplemented with DNAse I (10 g/mL) and EDTA-free protease-inhibitor cocktail (Roche). The mixture was sonicated for 15 min (Branson Sonifier 250, 1/2-inch flat tip, output 6, duty cycle 50%). After stirring the extract for another 40 min, the cell debris was removed by centrifugation (Beckman 70Ti rotor, 50,000 rpm, 1 h). The sample was passed through a 0.2-m filter and purified by immobilized metal affinity chromatography."


Expression of human peripheral cannabinoid receptor for structural studies, Alexei A. Yeliseev, Karen K. Wong, Olivier Soubias and Klaus Gawrisch
Protein Science (2005), 14:2638-2653

thanks for your answer!

80g is a really huge amount of cells. maybe this is exactly my problem. these receptors are expressed in low copy numbers. but I cannot use such amount of cells.

but I will check out this solubilization buffer

Jenny