Dear all, I am having problems with my Western blots. I am doing transient expression of proteins in Nicotiana benthamiana using Agrobacterium tumefaciens. These two proteins are both his-tagged (x10).After harvesting the tissue and processing it to get the extracts (+LB) I did the electrophoresis (SDS-PAGE) and the transfer to nitrocellulose membranes.
Firstly I incubated the membranes with anti-His (mouse), then anti-mouse-HRP and it didn't work, in the different plant extracts I could not see anything than background, not even a single non-especific band. I also tried using a different anti-His from other company and nothing changed. I know at least one protein is there because after incubating with the specific antibody against that protein, a big band appeared with the correct size.
We thought the problem may be due to a steric hindrance issue , so I also added 6M urea to the Laemmli buffer, and that did not improve the WB at all. I don't know what I can do. Can anyone give me some advice?
PD: sequences are correct, TGA after His tag. Positive controls were fine (same his tagged protein expressed in yeast). No proteolytic degradation after extraction, Coomasie gels showed neat bands.
Thank you very much for your time and help.
This recently happened to us, we detected our his-tagged protein with its specific antibody but nothing with anti-his antibody while we knew our anti-his antibody works well, so after some checkings, we concluded that the his tail wasn't enough accessible for the antibody. Finally We were surprised to see that we could still purify it on cobalt beads.
Hi,
Make sure that your protein is expressed by using a positive control for another protein detected previously. If you are sure that your protein is there then maybe the his tag is not accessible for the antibody binding.
Please check the fowllowing link to troubleshot your problem.
http://www.biotechniques.com/multimedia/archive/00043/BTN_A_000113147_O_43049a.pdf
Good luck
Hi,
There can be many reasons,
1. Make sure you are making your sandwich for blotting nicely by eliminating all big and small bubbles in sandwich. This can be problem sometimes and you see nothing in WB
2. You can change antibody, it is a possibility that antibodies are not good so you can change them.
3. Sometimes changing location of tag can also be a good idea, in my case i was not getting good protein expressions so i changed N-Term-his tag to C terminal.
Good luck
Can you clarify that you have run both coomassie and WB on the samples and while you see the expected band size of your protein of interest in coomassie, you don't on WB when you detect using anti-His?
Also is there any difference between the construct you put into yeast (your pos control) and the construct you put into Nicotiana benthamiana?
The protein could be undergoing post-translational modification in that the his-tag is cleaved - it sounds like you've C-terminal tagged the protein since you said the stop is after the his? Try putting it on the N-term with ATG upstream of his-tag and remove ATG from the coding sequence of the protein of interest?
Unlikely to be steric hindrance as SDS-PAGE is denaturing so by adding Urea it's unlikely to do anything more (apart from being a stronger denaturant than SDS).
Hi,
Have you check whether your protein of intrest is their in SDS-PAGE gel. If yes after membrane transfer you saw the same in the membrane by Ponceau stain. If yes have you added positive control only His tag protein.
Since His Tag is very small protein their is chance to escape. Check the possibilities and try with PVDF 0.2.
All the best.
check every steps
1. after membrane transfer, remaining gel stain to check the whether it is transferred completely or not
2. might be antibody is not working
This recently happened to us, we detected our his-tagged protein with its specific antibody but nothing with anti-his antibody while we knew our anti-his antibody works well, so after some checkings, we concluded that the his tail wasn't enough accessible for the antibody. Finally We were surprised to see that we could still purify it on cobalt beads.
Hi
Xi Jiang, in your case, we better check with our protein specific antibody. Even then it does not detect you have to think about its expression in host. If it is detected then better go for purify as Malika Moussouni does(to make sure your expression was there or not). If not expressed 1) N-terminal tagging, 2) using other vector for expression are preferred.
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