I want to pull down a membrane bound cytoplasmic GFP tagged protein. For this I ordered GFP tagged beads from chromotech. I am lysing the cells expressing GFP tagged protein of my interest with lysis buffer (150mM NaCl, 1mM EDTA, 1% Triton X-100, 50mM Tris-HClPh 7.4). After that i incubate the protein lysate and the beads tagged with ab for GFP at 4 degree for overnight. Next day i give it 3 washing (washing buffer : 250mM NaCl, 1mM EDTA, 2% Triton X-100, 50mM Tris-HCl Ph 7.4) to get rid of unbounded protein. Finally i boil the beads for 10 min at 90 degree with 2x sample loading buffer with 4% mercaptoethanol and separate the proteins in 10 SDS PAGE. But I am unable to see a good intensity band that i can send it for MS. Even i pull down cells to increase the band intensity but there in no improvement.
You might try the following: use 0.5% NP-40 as detergent instead of Triton X100 (it should interfere less with the ab-binding), 2nd: for the washing - reduce the NaCl concentration to 150 mM (but not below) - that is the physiological ion strength - and might improve the binding to the ab.
Good luck, Johannes
Thanks Johannes, There is some improvement after replacing TritonX. Interestingly, when i saw pellet under the microscope after the lysis, still it contains a lot of fluorescence. That means my buffer is not able to take out my membrane bound protein. Science i am investigating the protein that is inter acting with my protein of interest i can not sonicate it.
can any one suggest how to get the membrane bound protein with harsh treatment?
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