overexpression and purif. rec. protein

[moviefreak]

ok, here's a lab question, hopefully somebody can help me out.

I tried purifying an overexpressed recombinant protein(a protein of interest fused with GST which binds glutathione) using glutathione beads. I ran this sample on SDS-PAGE and performed coomassie staining. I also performed Western blot on an aliquot of the same sample. However, the results were negative for coomassie staining and Western (like there were no bands at all, clean as a slate). The molecular markers in both worked fine. Positive control only worked for coomassie.
Here's the catch, from the same sample, I aliquoted some for quantification of protein concentration (OD sample) and there were proteins in the sample.
How come I can't detect this protein in coomassie and western when optical density says there is protein in the sample?

some insights?

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[Angeliqua]

The OD reading is not specific to your protein, it is picking up total protein concentration, basically anything that can affect the OD. Did you add sufficient protease inhibitors THROUGHOUT the purification? ie. in all of the lysis, wash and elution buffers? If not, than it is possible the protein was degraded and the OD is picking up the tiny bits of protein that are too small to show up on the gel or blot.

 

[arsh]

There could be a lot of things going on. One possibility is that there was protein degradation. Did you add protease inhibitors at the proper steps? Also, ODs can be notoriously misleading. It is possible that the glutathione that you eluted with oxidized. Oxidized glutathione can result in an OD. There could also be some weird contaminant. Honestly, stuff like this happens all the time - that's just the way research is. I am sure you'll figure it out though.

 

[arsh]

haha! angeliqua and i were both thinking PIs.

 

[Angeliqua]

Also, what were you purifying the protein from, Sf9 (insect) cells, bacterial cells, mammalian cell lysates, etc. I work on a protein that gets "trapped" into all of the above mentioned cell pellets after lysis because it is part of a complex that ( as I now know) requires aggressive lysing. Try a different lysis procedure, add some (or more) protease inhibitors. Good luck.

 

[Angeliqua]

Originally posted by arsh
haha! angeliqua and i were both thinking PIs.

Anyone else have any ideas?

 

[mvervaine]

if you aren't getting any proteins stained with coommassie, something must be up. i've had this problem with coommassie, maybe you should try silver staining (from biorad. it's easy and no mess involved). it picks up some background but is about a gazillion times more sensitive.

as for the fact that your westerns are coming up blank, this could mean your detection process is wonky. play around with antibody concentrations, and maybe expose the film or (if chromagenic) stain it in the alkaline phosphatase solxn a little longer. either way, controls are very important in protein staining/blotting. if even your controls aren't working (and you are sure there are proteins present), that means the problem is probably with the staining/detection process, and not with the proteins themselves.

 

[Necrotic]

Hi... I apologize for the long, mostly technical response.

(I'm assuming you've have positive controls with you're westerns... if not, may be the antibody.)

First, OD at 280 is a direct reflection of the number of aromatic residues present... ie, W, F, and Y residues... mostly the tryps, though. Thats why an A280 is sometimes misleading, because the number of these residues vary. Also, these residues may be there, but there's no peptide bonds... this is why bradford assays are better for protein quantifications.

One thing you might want to try is taking the resin from your column after you ran the prep, boil it in SDS buffer for a few minutes (3-5), and load that directly on a SDS-PAGE (with proper control). If you see somthing there, then your elution step isn't working... if you don't, then you're not expressing the tagged protein or your beads aren't working.

Also, make sure that there are no frame-shifts between the tag and the target. If its a C-terminal tag, check that you eliminated the stop codon between your target and tag.

Another thing, it may be that your protein is either toxic or insoluable. To check for toxicity, do a growth curve with induced and uninduced cells (assuming its bacterial expression system, though could be done with other expression systems). To check for soluablity, collect samples at every step of the prep... pre-induced, induced, spun-down, etc... and run on coomassie/western. If this is it, it may be worth trying other N-terminal tags, some which help soluability... my favorite is the Novagen INTEIN system (I'm NOT a rep for Novagen).

Finally, do you have any weird sequence abnormalities? I had a large poly-Q repeat that for some reason or other completely eleminated expression... no idea why, but when I cut it off, it worked fine. Look at the sequence and structure (if possible), and see if anything sticks out.

Hope it helps. Don't give up.
N

 

[moviefreak]

thank you all for responding

i think it must have been the proteases degrading my protein since i didn't add any inhibitor and was working a tad slow. i work in a dna/rna lab and didn't know a lot about proteins. i appreciate all the comments and it will help me in my discussion of my experiment.

 

[DarkChild]

i agree with necrotic - the smells suspiciously like you're not expressing the protein you think you are. how are you expressing this protein? in vivo or in vitro?
are you sure you really have recombinant dna?
if I were you, I would go back to the DNA you used to transfect the cell line you used to express the protein or whatever. assuming you have primers for this stuff, run a PCR to check the size of your recombinant protein. did you have the recombinant DNA sequenced? if not, do so, you could have a frameshift. also, if you've transformed e.coli to grow up a bunch of your DNA, its possible that you could have had a deletion in that step.
(unless you're using the cells which grow up blue and white - sorry its been a long time since I've been in lab)
i suggest rerunning the gst binding column - who knows maybe you screwed up the protein transfer on the western. and its the easiest thing to check for.
then check your DNA to make sure you actually have insert.
good luck... let us know what happens though.