I have cloned a gene (1.52 Kb) in pQE-30 UA vector and transformed into E.coli M15 cells. The presence of insert was confirmed by sequencing. The protein was induced using IPTG and the over-expressed protein was analyzed on 12% SDS-PAGE under denaturating conditions. As compared to control, over-expressed protein band of approximately 48 kDa was observed in induced sample. But the molecular mass obtained in SDS-PAGE is not in accordance with that deduced by In silico analysis which suggests it to be of 58 kDa. Is it anomalous behavior of protein migration in SDS-PAGE. Can I assume it to be the protein of interest and proceed for further experiments. All kinds of suggestions will be appreciated.
It could be caused by proteases. So add as early as possible protease inhibitors into your sample preparation workflow. For both MW detection methods.
It could be also helpful to use different gels (from different manufacurers) or self made gels with different Gel-concentrations (linear gels are much better for a precise MW detection than gradient gels). Use also MW-standards from different sources.
@ Jager.............I believe it will be appropriate to confirm it by WB.
Anyway, thanks for nice suggestion.
10kDa reduction in size is not normal. First of all, if u have antibodies (also anti-6xHis should do) confirm in western that this is really your only (or the highest molecular weight) band for the protein. Sometimes you ll see faint band of expected size and this could be a major degradation products as Hluska has suggested above. Similar western test with a small batch purified protein will confirm this.
Good luck
Probably its degraded, or some kind of leader seq getting chopped off. But, as suggested above WB will give answer, or just sequence the band by MALDI
It could be caused by proteases. So add as early as possible protease inhibitors into your sample preparation workflow. For both MW detection methods.
It could be also helpful to use different gels (from different manufacurers) or self made gels with different Gel-concentrations (linear gels are much better for a precise MW detection than gradient gels). Use also MW-standards from different sources.
depends on the protein. The more hydrophobic, the faster it goes through a gel. Especially with membrane proteins you see them on much lower MW than expected (my protein is 66 kDa and the band appears at ~50 kDa). Try to rule out the proteases first, and then maybe use mass-spec to confirm you have the whole protein there? Tedious, but cannot think about anything else. It can be worth trying though, because maybe your protein undergoes some cleavage already in vivo... if it's a entirely new project, anything can happen, but if it is sth someone already tried and worked...
Did you check the integrity of your insert? Didi you clone by restriction enzymes or by amplifying by pcr? Can you exclude the presence of a premature stop codon in your clone gene?
You can try to quickly purify your target-protein and then run a LC/Ms to check its exact mass. Sometimes proteins do migrate anomalously.
As told, you may have some degradation. You can actually check if you have protolytic sites within your protein that will explain your results. Many software can do that, for example the GCG and EMBOSS suites. A google search should also return online servers to do that. In addition, if you run your samples less time or in a gel with a higher proportion of acrylamide (20%) you might see a lower band accounting for the missing MW. However, as also mentioned, it simply could be that your protein is not migrating properly. Make sure that you boil your samples long enough (up to 5`) and that you have a reducing agent on the loading buffer. If the problem persists and you can not see the lower MW band I told you about, then you could check the mass of your protein by MS. If you have purified protein you can check the mass directly (you may need to change the buffer). Otherwise, after SDS-PAGE transfer the protein to a PVDF membrane, cut the band and send it to the protein chemistry lab to determine the mass and the n-terminal amino acid sequence of your protein.
this is quite common when you are expressing some protein and getting a little lower size in SDS-PAGE. It could be due to aminoacid composition and complexity . It causes migration difference in SDS-PAGE. Need not worry and you can probe it with polyclonal or monoclonal sera or high titer of anti-sera of that particular pathogen expressing this important protein in western blotting for confirmations.
Best wishes
Raj
I would definitely check your protein's mobility under different conditions (non-denaturing) and see how the apparent MW compares to the one observed when the denaturing PAGE is carried out. Also you should be be able to use other techniques to estimate the MW of your protein, for example HPLC or MassSpec
Oh,
Just another comment similar to the one made by Alessandro Canella. The sequencing of the insert must be done thoroughly. It is not enough to just sequence it at the cloning junctions/ends but to carry out sequencing from both ends pass the entire insert at least twice or more until or discrepancies in sequence are resolved.
All the above suggestions are important, Sequence your inserted clone completely. confirm the protein on the gel with affinity purified protein specific antibody via western blotting. The protein separation on SDS-PAGE gels always give you 5 -10 kDa difference. Addition of protease inhibitors in samples will solve the problem.
In my article of 1993, I experienced a similar trend in SDS-PAGE and realized it was due to protease produced towards the end of the culture cycle. I did not use protease inhibitors. I washed the cells at a certain point and re-suspended in deionized water to get the products required intact
If your insert sequence is correct and in frame as discussed above, you could try a different expression strain. Is your gene of mammalian origin? If so, the strain you are using might not have enough of the rare codon tRNAs and expression might just stop when the cells run out, especially if your protein is relatively large. There are commercially available E. coli strains that encode for a number of rare codon genes like BL21's. BL21 strains are also deficient in two proteases as far as I know...Is the expression level normal or low in general?
As Tomasz said, some proteins will run at an aberrent apparent molecular weight in SDS PAGE gels. Another technique for determining the size is sucrose gradient density sedimentation. With proper calibration/controls this allows accurate size determination and can be performed if you do not have ready access to HPLC or Mass Spec facilities.
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