a similar situation happened to me. I ended up finding that the primary antibody I was using was not able to detect native protein while it was able to provide an extremely good signal of recombinant proteins. So if you can, I would suggest you to use a different specific primary antibody. It would be better if it comes from a different source.

Are you using Western blotting to confirm the expression?

@ Dr. Samadi, Yes i am using western blot to confirm the expression but not getting the band in it, but in SDS-PAGE i am getting well over-expressed protein in desired molecular weight...

Dear Gaurav, which object have you uised?  you should try first to get a positive control with antibody which for sure present in your smaples (actin, tubulin etc). Once you get a positive signal, you can use your protocols and chemicals fro overexpreed proteins.

Best wishes1

How did you confirm that there was overexpression? Did you use an antibody specific to your protein, but not His-tag specific or are you using a non-specific protein stain on the gel? What vector are you using to overexpress your protein and have you used the same vector before?

There are several reasons why the tag might not show up in WB, ranging from proteolysis by host proteases, to faulty cloning design. Also, the accessibility of His tags and hence the sensitivity of their immunodetection by anti-His mAbs varies -sometimes dramatically- according to the individual protein; see e.g. PMID 17081490.

Why don't you try capturing a little bit of protein via denaturing chromatography on Ni-NTA agarose? If the target protein shows up in the eluate then you'll have some circumstancial evidence that the tag is there...

Hope it helps!

mostly likely the Tag is not in Frame. also, seeing you overexpressed prot (I guess by coomassie staining) does not indicate that it is your protein, it can be another ORF.

other possibility you have a proteolytic site close to the Tag (since you still see the right apparent MW). But, I would expect the cleavage not being 100% of your prot. you can try running high percentage acrylamide to see if you have something at the size of the Tag MW.

Are you checked whether your protein transferred to the membrane or not.  Stain your membrane and conform the presence of desired size of your protein. So that you can come to an conclusion that your protein is degraded during the course of time. 

@Taras Pasternak, Hi I have used the other His Tag protein which i used several times as positive control. Even in this condition which i have mentioned in my question i am getting band for that positive control after western blotting but not getting the band for my other target protein.. 

Hi !! @ Jared Taylor,

I am getting nice over-expressed protein in comparison with the un-induced sample on SDS-PAGE results. Which might confirm that its my desired protein. I Cloned this protein in pQE30 vector and pET23a but same results in both clone..No band after western blotting..

Hi!! @ Alejandro Martin,

Thank you for the suggestions..i will look after your suggested points. I will try to purify it in denaturing conditions..But after using SDS and DTT or b-merceptoethanol also protein should get completely denature and its His tag should be exposed while performing western blotting... 

Hi, Gaurav, have you checked mRNA level with RT-PCR?

Hi !! @ Ceri A Fielding,

Protein is cloned in both vectors having N & C terminal. But no positive results in both condition..

Hi!! @ Yair Botbol,

I will try to confirm about the ORF with gene sequencing. But why every time its expressing in desired MW after induction and in compared to un-induced samples... 

Hi!! @ Shiyam Sundar Ramachandran

Proteins get transferred everytime. I confirmed it with Ponceau stain..and other protein which i am using as positive control also gives good signal..so i don't think there is any issue regarding transfer of proteins...

Hii!! @ Taras Pasternak

No i have not done that. I do not know about the real time PCR yet..That i have to look later..How it can help, can u describe??

Gaurav,

There is plenty of evidence out there demonstrating that Western blotted proteins are not all spread out, but retain or regain at least some of their structural features (perhaps during transfer). It is hard to find; as it is mostly mentioned in passing in papers dealing with other subjects, but is there. Bacterial porins seem to be particularly prone to this phenomenon. Also, look into the paper I pointed you to; many of the experiments are indeed Western blots.

And drawing from my own experience, I once generated a library of 74 Ala substitutions to study surface-exposed residues of a C-terminally His-tagged viral protein. Some (four or five) of the variants exhibited markedly decreased or increased reactivity to a control anti-His mAb in both ELISA and Western blotting, even though the proteins were intact as assessed by MS. I do not have any explanation for this phenomenon other than interaction of the tag with other residues of the protein, which necessarily requires a partially structured polypeptide chain.

It is little puzzling why you dont see signal when expressing either as an N or C terminal fusion. As you say you can see it clearly an expected size protein in SDS-PAGE/coomassie. There are couple of thoughts i.e. have you sequenced the cloned region so to confirm the reading frame at both N and C terminal...do you see a read through of transcript (check all three frames....may be expressing in different frame). Cleavage is  another concern as I experienced lately for a C terminal fusion protein. Try expressing a truncated version of the same protein...just a thought. good luck!!

1. Sequence your constructs to make sure they are in right reading frame to express with His-tag.

2. We routinely run the lysates of uninduced and induced  cells (pellet cells from 0.2ml [uninduced] or 0.1ml [induced] of culture and biol in 0.1ml of 1X SDS-PAGE buffer)  on SDS-PAGE and stain with coomassie blue to visualise the expression of desired protein (X protein) band only in induced cell lane. Off course, you may not see the X protein in coomassive blue stained gel if the expression is low.

3. Then you try to do immunoblotting of whole cell lysate using anti-His antibody and  X  protein anitbody .

If you see the X protein as a prominent band only in induced cells lysates loaded lane in coomassive blue stained gel and by immuoblotting with X protein antibody but not by immunoblotting with anti-his antibody, then it may indicate loss of His-tag from X-protein due to some reason.

You should definitely check mRNA with qPCR. Is there an antibody specific to your protein that you could use instead? I had a similar problem in the past with a FLAG tag. My protein was over expressed as confirmed with protein specific antibody, but I could not detect it with the FLAG antibody. This was despite having checked to see if it was in frame and using denaturing conditions.

a similar situation happened to me. I ended up finding that the primary antibody I was using was not able to detect native protein while it was able to provide an extremely good signal of recombinant proteins. So if you can, I would suggest you to use a different specific primary antibody. It would be better if it comes from a different source.

@ Diane Tshikudi: Thank you for the advice. You have suggested about the primary antibody. In my case I have positive control with same tag to my target protein. After western blotting primary antibody is able to detect the positive his tag control but not the other target protein why? In my case there is no such antibody available for the protein so i am confirming it with anti- His tag antibody.. need some more disscussion..its a common trouble which researcher are facing now a days..

Either u can check with antibody specific to your desired protein or just check your reverse primer used for cloning whether it results to His tag out of frame

Amit is correct. Never bet on one horse. You need both a good working His-specific antibody and in addition a good antibody specific to the protein of interest. They both should confirm each other by comparing control and over-expressed lysates.

I agree with Amit Deshmukh, It seems like you have your His-tag is out of frame, because this will give you a protein with the same molecular mass in SDS-PAGE. Another possibility is that your His-tag in C-terminal can be hiding inside the folding.

Dear researcher

can you see link:

http://info.gbiosciences.com/blog/bid/130848/7-Common-Problems-with-Western-Blotting-Solved.

7 Common Problems with Western Blotting Solved

Regards