Blotting a nuclear protein and nuclear protein extractions in tissue

[Pete_2007]

Hi all,

This is my first post, and when I saw the great help offered by many for troubleshooting westerns, I knew I had to join!

Ok, so my problem, in short, is as follows: I've begun working as a grad student doing an MSc in a mouse genetics lab, and the first part of my project involves some biochemistry, including western blotting on a nuclear fusion protein to beta-gal. This same fusion protein has been detected in mouse tissues using Beta-gal staining.

However, when I have tried to blot to detect this protein in protein extracts from the same tissues, we cannot detect it at all. I have optimized the blotting protocol, including dilutions of 1o and 2o antibodies. The protocol works on E.coli lysates expressing Beta-gal - which is the positive control. So I'm confident that if that protein is there at ~>0.1 ng (the average lower detection limit of a blot), I should be able to see it. Any ideas on this?

Furthermore, I have been trying to prepare nuclear protein extracts from mouse tissues, which has become a pretty arduous task! Our lab isn't really setup for too complicated protein work. Nevertheless, I would appreciate any ideas as to protocols or products anyone has used for preparing nuclear extracts from tissue.

Peter

[danfive]

Are you running at native or denatured protein conditions? Consider comparing the two.

Are you comparing mouse tissue lysates to control E.coli lysates? Is you E.coli control making a recombinant protein?
Then perhaps a similar recombinant protein was also used to make the antibodies....you may want to look for polyclonal Ab to boost your chances....

[Pete_2007]

hi Dan five, thanks for your reply, to clarify:

1) I am running SDS-PAGE in sample buffer with DTT, so denaturing and reducing conditions.

2) I am trying to detect a fusion protein - the N-terminal region is our protein of interest, while the C-terminal region is Beta-galactosidase. We can detect this fusion protein by beta-gal staining in certain tissues, but using an anti-beta gal 1o antibody I cannot detect the presence of this fusion protein in the same tissues.

3) The E.coli cell lysates are positive controls for our anti-beta gal antibody, because they are from cells that were expressing huge quantities of beta-gal prior to lysis, and so blotting this extract shows a nice clean band at ~120 kD (as expected for beta gal). So no they are not expressing our fusion protein.

Any further ideas would be very appreciated!

Peter

[danfive]

1. OK. Reduced and denatured

2. So that proves that the reporter gene and its product work in vivo, but the reduced and denatured product is unrecognized on WB. My suspicion is the sample preparation, i.e. protein extraction. I assume the E.coli and mouse tissue have separate optimal protein extraction procedures.

3. So the control should and does work well. Since this is a fusion protein it will be bigger than 120kD (right?) so another thing will be that the actual gel electrophoresis and blotting for such a big protein can be a problem.
You should look for protocols especially for WB on large proteins; should include running the right gradient gel at low voltage and possibly using methanol (at low conc) in the running or transfer buffer; and maybe even changing from tank transfer method to semi-dry transfer (or just optimization for either). ---> westernblotting.org - Troubleshooting Western Blot Results

[Pete_2007]

Sounds good. I hadn't considered the size being such an issue with the transfer, but that would make sense. I had also considered the protein extraction being a problem. I was preparing whole cell extracts, but because this is a presumably low abundance transcription factor, it would be a good idea to enrich it by only looking in nuclear extracts, hence my problem with getting a good one of these.

We do a submerged tank transfer onto PVDF, with cold Tris-glycine buffer + 20% methanol. I will try to transfer for longer times I suppose this may help.

Peter

[danfive]

Originally Posted by Pete_2007

Sounds good. I hadn't considered the size being such an issue with the transfer, but that would make sense. I had also considered the protein extraction being a problem. I was preparing whole cell extracts, but because this is a presumably low abundance transcription factor, it would be a good idea to enrich it by only looking in nuclear extracts, hence my problem with getting a good one of these.

We do a submerged tank transfer onto PVDF, with cold Tris-glycine buffer + 20% methanol. I will try to transfer for longer times I suppose this may help.

Peter

I hope you mean Tris-glycine-SDS +methanol for transfer. If not you may want to start by adjusting that as well.
You can also enrich by filtering through a molecular weight cutoff spin filter (check out Microcon MWCO filters).

[Pete_2007]

We have never used SDS in our transfer buffers. What concentration should it be at? 0.1% or something? I will try that next time.

MWCO spin filters are a great idea, thanks!

Peter

[danfive]

Yeah SDS at 0.05-0.1% final concentration.

[celina]

Hi Peter and Danfive:
I am having the very same problems as Peter has (or I hope, had): I expressed a recombinant protein in E. coli (a nuclear protein from the plant A. thaliana), generated rabbit antibodies against it and then prooved they specifically recognized the recombinant protein. However, when I do Western Bolts of Arabidopsis nuclear extracts I do not get a signal, although the control is ok. We´ve even tried chimioluminiscent western blots, with no result.
What happened in the end with your experience? Any suggestions?
Many thanks, hope to get a reply!

[danfive]

Originally Posted by celina

Hi Peter and Danfive:
I am having the very same problems as Peter has (or I hope, had): I expressed a recombinant protein in E. coli (a nuclear protein from the plant A. thaliana), generated rabbit antibodies against it and then prooved they specifically recognized the recombinant protein. However, when I do Western Bolts of Arabidopsis nuclear extracts I do not get a signal, although the control is ok. We´ve even tried chimioluminiscent western blots, with no result.
What happened in the end with your experience? Any suggestions?
Many thanks, hope to get a reply!

1st. Is extraction technique effective? CTAB, Kit method?
2nd. Is protein rare or minimally expressed? may consider enriching for the rare protein (like the MWCO) or 2D gel+WB to maximize the variety and amount of proteins transferred to gel and subsequently blotted to membrane. (obviously one 2d gel is for your positive control, then separate one is sample, etc)
3rd. what is the protein size? large, small?