Dear fellow researchers,
I was wondering if different forms and stages of post-translational modifications (such as N-linked glycosylation) on a recombinant protein expressed in insect cells can be responsible for various thick bands observed post affinity purification* in my SDS-PAGE gel? I would need the unknown protein (predicted at ~55kD and possible glycoprotein) for interaction assays.
*Binding and wash buffer was 100mM Imidazole, 20mM Sodium Phosphates, 0.5M NaCl and 0.1% Tween-20. Elution was done with 500mM Imidazole and all the other components listed above. HisTrap 5ml column used.
I would be happy for any advice, opinion or past-experience.
Thanks,
Julian
Hi Julian Ramelow . A general rule of thumb is that heavily glycosylated proteins appear "fuzzy" or diffuse on SDS-PAGE. Plus, glycosylation rarely adds more than ~10% to a protein MW on SDS-PAGE.
I don't understand the rest of your post.
A likely explanation for the multiple bands is that the metal affinity chromatography was not sufficient to purify the protein to homogeneity, and the other bands are contaminating proteins.
You could check this by Western blot using an antibody specific to your protein.
As Steingrimur Stefansson pointed out, glycoproteins have fuzzy bands in SDS-PAGE. This is due to variable numbers of negatively charged sugar-residues. If you need crisp bands, perform electrophoresis with a positively charged detergent (CTAB) instead (DOI:10.1016/S0003-2697(02)00639-5, DOI:10.1007/978-1-59745-542-8\_14)
You purified via the His-tag right? I assume that because of the imidazole in your buffer.
The His-tag may lead to high protein yields, but its purifications can be quite unspecific, especially if you use Ni-NTA products instead of Co-NTA.
So either change the tag to something more specific like GST or Strep (FLAG and RHo1D4 might be too much), or you must follow up with a Size Exclusion Chromatography.
As Adam B Shapiro point out, you probably have contaminant proteins in your purification. You can also use an anti-His antibody in a Western blot assay if you don't have one specific for your protein. Good luck!
Dahlia A. Awwad
: That may be true for O-linked carbohydrate, N-linked carbohydrate can add several 105 Da. For example, the multidrug-resistance transporter 1 (Mdr1, Pgp, ABCB1) has a protein mass (from sequence) of 141 kDa, but runs to about 170 kDa because of N-linked sugar trees.
Thank you everyone for the helpful answers. I tried Size Exclusion Chromatography and it solved the contaminating protein issue.
Hello everyone,
I wanted to post another issue that I am facing that has to do with this post. I tried a Western Blot which was recommended by Pier Paolo D'Avino and Adam B Shapiro I used my gene specific pAb. However I am getting three bands (see image). I am using the baculovirus system and purification procedure as above. Samples were heated at 95*C for 5-10 min and I used BME as reducing agent. I also used a virus infected unrelated protein and uninfected high five lysate as controls.
What could be a reason for this and how can I interpret this? I am really clueless and unsure what it means for my protein. Should I try with DTT as reducing agent? Thanks for any advice or help.
DTT is the better reducing agent, but I'd not expect it to help in this case, which looks like proteolysis to me. Try to lyse your cells directly in sample buffer and blot this, this should tell you whether the degradation already occurs in the cell or only later during purification. In the latter case, you need to find out which protease inhibitor works. The former case would be harder to deal with.
I would like to know the masses of the standards on the gel in order to see whether it is possible that the 3 bands correspond to the expected mass and 2 and 3 times the expected mass. I have seen that pattern with a protein, despite using denaturing conditions and reducing agent.
Adam B Shapiro thank you for the response. If you click on the image you should be able to see the markers. Starting from the top it is 180, 130, 100, 70 (Orange), 55, 40, 35, and 25 kD. Based on my calculation there does seem to be a match…my protein is ~55kD. Now, I am highly curious as to what is your reasoning behind this? What proteins show this behavior?
A protein I am working with at the moment shows this behavior on some gels with fractions from an IMAC column. I can't tell you which protein it is. I don't know why it happens. It isn't caused by an absence of reducing agent. Maybe someone else on RG can supply more information.
Adam B Shapiro Engelbert Buxbaum I was doing some reading and could it be that the protein is ubiquitin tagged? I read it adds about 9kD to the apparent weight.
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