Western blotting for membrane bounded protein - (Dec/14/2007 )
Hi all,
I am trying to do western blotting for a V5-HIS tagged recombinant protein of Fatty acyl reducatse, which is membrane bounded and I failed to see them on blot. I am using insect glow system (invitrogen) for expression in SF9 cells. My protocol as follows:
After transfection, 2 days incubation & confirmation the GFP expression; scrape the cells with lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris pH 7.8) and then vortex cells at 4oC for 10 min to ensure they completely lysed. After centrifugation at max speed, lysate and cells pellets were mixed with 4x SDS sample buffer and boil for 5 min and load on the gel. But after blotting, I failed detect the protein. Please teach me how to do western blot with membrane proteins?
Thanks
Binu
the np-40 of your lysis buffer may have interfered with the binding of sds to the proteins. you may have to dialyze the samples against buffer without np-40 before adding the sds buffer.
I am trying to do western blotting for a V5-HIS tagged recombinant protein of Fatty acyl reducatse, which is membrane bounded and I failed to see them on blot. I am using insect glow system (invitrogen) for expression in SF9 cells. My protocol as follows:
After transfection, 2 days incubation & confirmation the GFP expression; scrape the cells with lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris pH 7.8) and then vortex cells at 4oC for 10 min to ensure they completely lysed. After centrifugation at max speed, lysate and cells pellets were mixed with 4x SDS sample buffer and boil for 5 min and load on the gel. But after blotting, I failed detect the protein. Please teach me how to do western blot with membrane proteins?
Thanks
Binu
I've been reading a bit about doing westerns of membrane proteins, and many protocols suggest that you don't boil your samples as this can cause aggregation of proteins. They recommend incubating at 37C for 15-30 min. Also, do you know that your antibody is working? From what you've written it sounds like you can use a GFP antibody. Is this what you're using, and can you confirm that it works on other samples?
NP-40 is a pretty soft detergent. Maybe the membrane embedded proteins are not solubilized. I suggest you try out with Triton-x100 at 1% final concentration. The membranes should then be solubilized.
Yeah! I also tried 1% triton x100 in the lysis buffer. till now I havent succeded. Later I changed the protocol like this: After harvesting the cells in the lysis buffer (50 mM Tris pH 7.8; 150 mM NaCl, 1 % NP 40 & 1% Triton X 100) centrifuge & remove the lyasate into a new tube. Cell pellet dissolved in 50 mM Tris pH 7.8, 150 mM NaCl, 0.5 % CHAPS and prot. inhibitor. Incubated the sample at 4oC for 2h in a rotator. Thereafter, mixed the sample with 4x SDS buffer and incubated at 37oC for 1h. After incubation, sample loaded on a gel & did western. But when I checked, no band!
I am trying to do western blotting for a V5-HIS tagged recombinant protein of Fatty acyl reducatse, which is membrane bounded and I failed to see them on blot. I am using insect glow system (invitrogen) for expression in SF9 cells. My protocol as follows:
After transfection, 2 days incubation & confirmation the GFP expression; scrape the cells with lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris pH 7.8) and then vortex cells at 4oC for 10 min to ensure they completely lysed. After centrifugation at max speed, lysate and cells pellets were mixed with 4x SDS sample buffer and boil for 5 min and load on the gel. But after blotting, I failed detect the protein. Please teach me how to do western blot with membrane proteins?
Thanks
Binu
I've been reading a bit about doing westerns of membrane proteins, and many protocols suggest that you don't boil your samples as this can cause aggregation of proteins. They recommend incubating at 37C for 15-30 min. Also, do you know that your antibody is working? From what you've written it sounds like you can use a GFP antibody. Is this what you're using, and can you confirm that it works on other samples?
This time I did not boil the sample. after mixing with SDS sample buffer, I incubated the sample at 37oC for 1h.
I am using V5 epitope Ab. This Ab is working with positive control (pIZt/V5-His/CAT) & I got 34 kDA band (CAT protein), but this is not a membrane protein.
After transfection, I checked efficiency using flurescence (GFP) & selected the stable cell line by zeocin (antibiotic) as a selective agent. Expression vector, pIZt/V5-His has 2 promoters; OpIE2 for expression of gene of interest and OpIE1 for expression zeocin and GFP for detection of transfected cells and selection of stable cell lines. (more details refer http://www.invitrogen.com/content/sfs/manu...elpizt_man.pdf)
OK. let me try this way.
How about harvest your cells directly in 2x (or 1x) SDS loading buffer (the Laemmli buffer)?