I attempted to run:
Goal: To test binding between a protein in its native conformation and a monoclonal antibody that shows cross-reactivity with this protein in its denatured form (determined by SDS-PAGE)
Experiment: Blue native PAGE Western blot, 3-8% Tris-Acetate gel, ran at 130V for 90 mins, transferred at 30V for 60 mins, reagents include anode/cathode buffers (from 0.5M MOPS and 1M Tris-base stocks), 1X loading buffer (Coomassie Brilliant Blue R-250).
Issue: The stain of only the experimental protein (not other protein as controls) remains after the transferring from gel onto PVDF membrane. This is before primary incubation and developing.
Lessons: The stain remaining issue is not due to high concentration of the protein loaded.
I would highly appreciate your ideas and suggestions to troubleshoot this issue!
Hi there,
You may avoid Coomassie blue in the loading buffer if protein staining is not required. Here a recipe from Biorad :
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006027F.pdf
Hello Dominique,
I will try doing that. It will definitely resolve the issue, but do you have any suggestions to determine whether the proteins are running okay in the gel or whether they are transferred onto the membrane?
If they run OK when Coomassie stained they should also run OK in absence of Coomassie.You may use Ponceau Red for staining the membrane prior to probing to check transfer efficiency (https://pages.wustl.edu/levin/ponceau-staining).
Thank you so much for the suggestions. I will try it next week. Have a good weekend.
So I tried running the gel again with everything the same except with no Coomassie blue r250 added to the 1X loading buffer and the cathode buffer. The stain issue obviously is resolved but then the western blot failed. Although I didn't stain the gel with Ponceau prior to transferring to confirm if the proteins are there, it could be the protein are there but not transferred or no protein was able to run through the gel without the dye (because I thought I have read somewhere that R250 dye actually also needed to attach the charge for the native protein so it could be ran in blue native gel). Any suggestions how I could proceed? Would changing PDVF into nitrocellulose membrane, trying Coomassie Blue G instead of R-250, etc.?
Have you tried destaining the membrane in methanol after transfer but before blocking and incubation with primary antibody? This eliminates background CBB stain on the blot. When I tried this, the proteins remained stained, but the western blotting still worked for the particular protein that I was interested in.
A word of warning... I just tried Lisa's suggestion (after reading similar here http://www.abcam.com/protocols/blue-native-electrophoresis-protocol) - but the methanol started dissolving my blot membrane and ruined it. So I guess this can't be done for nitrocellulose, maybe only PVDF?!
As stated on page 29 of the user guide for the NativePAGE Novex Bis-Tris gel system from Thermo Fisher, " PVDF is the recommended blotting membrane for western blotting with NativePAGE Gels. Nitrocellulose is not compatible for blotting NativePAGE Gels since the nitrocellulose membrane binds the Coomassie G-250 dye very tightly and is not compatible with alcohol-containing solutions used to destain the membrane and fix the proteins." https://tools.thermofisher.com/content/sfs/manuals/nativepage_man.pdf
I would go with Lisa's suggestion to destain with methanol. The only downside is that the molecular weight marker isnt visible anymore.
To do that, I also included a lane just with the marker on the gel which i cut out, fixed and stained with coomassie (if need be). Then use this as the ladder by aligning the gel and the blot.
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