I am trying to perform Western blots for HIF-1alpha. I have so far had one successful attempt using a nitrocellulose membrane. When I attempt to perform the same lysis protocol, gel running conditions and transfer onto a PVDF membrane, I only get detection in my CoCl2 treated HeLa positive control sample, but get it in my positive control as well as all my samples w/ nitrocellulose. Both 0.45uM pore size.
My transfer buffer contains 25mM Tris Base, 192mM glycine, 0.1% SDS, 20% Methanol
Has anyone had the experience where HIF-1a (or any protein for that matter) transfers better onto nitrocellulose membrane as opposed to PVDF? Would prefer PVDF as we have plenty of it in the lab and would have to order nitroceullose, and so I can strip and re probe for other proteins if I can get to work with PVDF.
Thanks!
Hi Sean,
if anything, PVDF should work better. Did you prewet the membrane with methanol or ethanol before transfer as this is critical?
Cheers
I did western blot expression of HIF alpha in animal tissue homeganates, In my case i used PVDF membrane and results were good. Nitrocellulose membrane is good when the bands you get are strong, for light bands its difficult to detect. PVDF is better in those cases. Try to activate your membrane with methanol before transfer. Hope you get good results.
GoodLuck
Skip SDS from transfer buffer. SDS reduce protein binding with membranes.
HIF-1 alpha expression is very low in basal condition, as you know, hypoxia induced its expression, and its half-life is very short too. I did the similar experiment as you, the CoCl2 treated cells had dramatic expression of HIF-1 alpha, while the control cells had very weak expression, almost undetectable. I used the PVDF, and no SDS in the transfer buffer.
Hi,
PVDF membrane is better for proteins transfer, stripping and reprobing .
Wet transfer buffer comp: 14.4 g of Glycine, 3 g of tris base dissolve in 800 ml of Milli Q water and adjust pH to 8 then add 200 ml of methanol. keep in 4 degree Temp.
Wet transfer at 4 degree temp, 100 V for 2 hrs is better for sensitive proteins.
PVDF for sure. Works the best. I usually soak the PVDF membrabe in methanol for 10 min prior to transfer. Also you need to optimize your antibody conditions and amount of protein to be loaded in gel. I used about 40ug of protein and it worked.
Hey Gravel, I use the HIF-1alpha antibody from BD Biosciences. Have read that BD or Novus antibodies for HIF-1 are typically the best?
PVDF is as good as nitrocellulose. The protein binding works via hydrophobic interactions on both kinds of membranes. Remove the SDS from your transfer buffer, this will inhibit for both membranes hydrophobic interactions.
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