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HELP!?!? with MEMBRANE PROTEIN extraction - Extraction of GPCRs from HEK293T for IB and IP (Feb/20/2011 )

I have been struggling with the extraction of several GPCRs in HEK293T cells. The major trouble is that although the receptor specific antibodies can recognize the protein specifically because lanes from cells not expressing the protein are clean, there are many bands. The major bands appear around the expected MW but there are so many other bands. I thought that it was because of glycosilation but PNGase f treatment did not clean it up. I also tried including a strong reducing agent Iodoacetamide it helped a little. I also have tried several extraction methods from total cell lysate to membrane fractionation. My GPCRs are tagged but the tagged antibody condition is not nearly as sensitive as the receptor specific antibody. Currently, I think the extra bands must be due to aggregation because I found previously that sometime the receptors could not enter the gel and remained in the stacking portion.

Has anyone else experienced this kind of trouble with membrane protein extraction from mammalian cells?

Do you have any tips to reduce the aggregations?

My current protocol
Plate cells at 3.5x10^6 on 10cm
24 hours later, transfect with 2ug total plasmid DNA
24 hours later wash cells in PBS on ice
Collect with rubber policeman in homogenization buffer (50mM Tris HCl pH7.5, 1mM EDTA, 150mM NaCl, 1%NP-40)
Cell lysis with 25G needle (sonicate 5" x1)on ice
cent at 3000rpm to remove nuclear pellet at 4C
either proceed to ultracentifuge for membrane extraction or take sup to new tube and measure protein concentration
Add 1x SDS buffer with 50mM DTT and heat to 37C for 30'
Load onto SDS PAGE gel

I have been optimizing with other detergents to try and find a suitable IP buffer

I attached some sample blot which had also been treated with stimulation by the receptors agonist .

All suggestion welcome :)

I have been working the kinks out of this for over 1 year!! :o I just hope to succeed at seeing a reasonably distinct band at expected molecular weights...

Thanks in advance

-Aliza Ehrlich-

Hi there, i do work with the G-protein, so usually i use TE buffer 150mM NaCL with a protease inhibitor containing NO detergent, then homogenize with a polytron and centrifuge at 600 g for 10 minutes 4c, then Ultra cenntrifuge at 100000 g for 30 minutes, resuspend the pellet in TE buffer containing 1% triton X100 and shake at 1400rpm (eppendorf mixer 4c), centrifuge at 16000g for 10 minutes, and then you have your membrane fraction, now in case of IP instead of using 1% triton use 0,1% and to homogenize use polytron, you can use it directly for IP. i hope that helps, if you need anything else just say !