Western for membrane bound protein - (Nov/27/2007 )
Hi all
I am trying to set up western blots for my protein which is membrane associated and have failed to see them on blot with my regular lysis buffer (1% NP40, 150 mM NaCl, 20 mM Tris)
Do I need to change the lysis buffer, if so then what should I go for? I need to extract my protein from the RAW cells.
Rits
Did u try first purification under denaturing condition, and testing on acrylamid gel? can u use denaturing buffer?
How do u isolate ur protein from the membrane? do u use lysozyme and sonication befor purification?
try SDS buffer for extraction and sonicate the sample adequately.
How do u isolate ur protein from the membrane? do u use lysozyme and sonication befor purification?
No I can not use the danturing conditions for my experiments unfortunately. I am simply trying to lyse the RAW cells which are expressing this protein (transfected stable cell lines confirmed by FACS and microscopy) and use the whole cell lysate for my western blots using the bugger I mentioned. But it does not seem to be working and I assume that the lysis buffer is not good enough to broing the protein out in the extract. Need help!!!!
Like scolix said, if you just want to see expression of protein in cells, just scrape them in SDS sample buffer (aka loading buffer) and then load gel directly, followed by WB. If not, you can try to add more detergents and/or stronger detergents into your lysis buffer. Try 1% TritonX 100 first, then see if 0.1%SDS could help.
I don't understand why you can't use denaturing conditions, though. According to what you wrote, you only want to see the protein expression in cells?
I don't understand why you can't use denaturing conditions, though. According to what you wrote, you only want to see the protein expression in cells?
Not really, I need to do IPs after I can see my protein on western blot and using SDS could damage the protein theerfore won't be able to use this. Do need to solubilise my protein so I can make the western work and then will be doing IPs :-(
As Almasy suggested try triton for extraction and sonicate adequately.
Hello rits
U should look for protocols " purification of membrane proteins" (pierce)
or look for suitiable reagent like celLytic-Y by sigma
i only have old protocol i never try:
resuspend the cell pellet i 50mM Tris pH 8; 100mM KCl + protease inhibitor
adding "decyl-beta-maltoside (DM) at 120mM. sonication and incubation at 4C for 3h with agitation
centrifuge at 14000 for 5 min at RT and proceed for analysing
bye
Low amount of SDS is not necessarily damaged the interaction of proteins. Certainly, in certain cases, SDS could cause problem, but it is not always so. RIPA buffer is quite a common one used for IP (just look at its name), and there is SDS in that (or at least in the modifed ones, or is this the modified RIPA won't have SDS? Anyone, help?). So if the milder detergents like Triton won't work, you may want to add in SDS, with very low concentration if you want (0.001 or 0.01%), and try. You never know if you don't try.
RIPA buffer is not good for membrane proteins. Use alternatives.
Temperature for denaturing is very crucial for some of membrane proteins. Have a trial using differernt temperatures (50-100 degree) to look if there is any difference, which might give you a surprise.