Hello Everyone,
What should I do to get rid of the RuBisCo band from Arabidopsis tissue sample?
1. My target protein was extracted from Arabidopsis tissue. I loaded 50 ug of protein. After transferring the gel to PVDF membrane, I did Ponceau S staining to confirm the transfer. There was a RuBisCo band along with another non-specific band after staining.
2. I kept blocking at RT for 2 hrs.
3. My protein was tagged by GFP and I used GFP monoclonal primary antibody (1: 5000 dilution), 4 degree overnight, and appropriate compatible secondary antibody-HRP (1:10000 dilution) for 2 hrs at RT.
I didn't get a band for my target protein, only getting the RuBisCO band after developing the blot.
Does anyone face this kind of problem or suggest me how to get rid of this?
Thank you in advance!
I was also looking for the answer to this question, facing the same problem with RuBisCo. But why nobody responded? :D
Try stripping the membrane in stripping buffer at 50C for 30min and after washing use the same blot for re-probing. Your rubisco will get rid. I am not sure, but I tried it once and it worked well with my blot.
If you find other solution please do let me know also.
I had the same problem and found the solution.
There is a very good antibody from Agrisera that does not give rubisco unspecific band. It´s the anti C-YFP antibody from Agrisera.
It recognizes only the C-terminal half of YFP, CFP or GFP and it works perfectly fine to detect the whole protein. I struggled a lot until I found this antibody and now doing WB is a pleasure (well... still tedious and all but gives nice bands :P). It gives No bands at all in the Col-0 control I always do.
https://www.agrisera.com/en/artiklar/c-yfp-c-terminal-of-yfp.html
check the blot to see how nice it works
As secondary I´m using anti-rabbit-HRP from santa cruz (a cheap one)
Hi Sreya Das
As per my experience with Rubsico and GFP-tagged proteins of similar size, I totally agree with Martiniano M Ricardi and would suggest changing the anti-GFP antibody.
In case your protein is a membrane protein, try using relevant extraction buffer and chloroform-methanol enrichment. This will surely help.
I think Himani Agrawal also has a valid suggestion worth trying.
All the best
Vivek
Vivek Dogra I tried it once, it worked but it is not consistent with rubisco So I switched to protamine sulfate precipitation of rubisco. So far it is consistent in removing larger subunit (53 kDa) of rubisco.
Sreya Das please go through these articles I guess it may be very helpful for you.
Article Time to dig deep into the plant proteome: A hunt for low-abu...
Article Depletion of RuBisCO Protein Using the Protamine Sulfate Pre...
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