Hello, I am working on a recombinant protein expressed in the form of inclusion bodies for my bachelor thesis. After solubilization and refolding I performed an SDS-PAGE and to confirm the identity of my protein I also performed a Western Blot. But when I compared the protein band on the SDS-PAGE gel with the band I obtained in the Western Blot, I saw that the band of my protein in the Western Blot was shifted upwards.
Does anyone have any idea what causes this upward shift? Logically, the band in the SDS-PAGE must be at the same level as the band in the Western Blot or not?
I hope that someone can help me.
Compare the SDS-PAGE band with the western blot band with respect to the protein ladder (marker) & see whether their kDa matches.
hi
1-Please provide your images with more details.( Protein size marker and ...)
2- Recombinant protein size kD ?
good luck
Hi:
I think your protein is with His tag, which will have a little bit higher MW.
Also, some modifications for the protein will occur in the E.coli.
So the band will be up shift.
for the WB, since the anti-his antibody will have some nonspecific band, you would add an empty E.coli as a control.
Hi Reza Hadavi Tushar Chakraborty and Junjie Shao
The size of my recombinant protein is 26 kDa. And I think in the gel of SDS PAGE the band has the right height. But in Western blot, this band is higher than the band in the gel of SDS PAGE. It is visible at about 30 to 32 kDa. I simply cannot explain this effect. I know that a His-tag can cause an upshift, but it should be visible in the gel of SDS PAGE as well as on the membrane of the Western blot, shouldn't it? Interestingly, I can also observe this effect if I use an antibody directed against a fragment of my protein instead of an anti-his antibody.
Thank you very much for your help and ideas.
Hi Irina,
I've had problems with gel inconsistencies in the past; specifically, nonuniform polymerization across the gel has lead to some confusing band shifts. If you are making gels from scratch, I would make sure your recipe solutions are fresh (especially APS if that's what you're using), give the pre-gel a really good mix and let the gels set longer than normal before loading.
If using preformed gels, complain and get some for free!
Alternatively, you might try to increase primary antibody incubation concentration (e.g. incubate in 1:1000 antibody:BSA instead of 1:2000) just in case the signal you're observing is non-specific.
Good luck!
Hi, Irina,
What kind of gel did you used?
I would use a pre-cast gradient gel (NuPAGE 4-12% with Mess buffer). Did you use the same amount of the sample for Western blot as for SDS gel? For me the signal from western is too low. I would check the identity of the protein per Mass spectrometry, if possible.
if they are the same protein from the same prep then there can logically only be certain reasons for this
1. protein was treated differently for WB i.e. reduced/non reduced. This would change the way a protein separates
2. your gels are not the same i.e. different % acrylamide, made at different times
3. You have run the gels differently i.e. one gel has been run further or faster
4. The markers are different i.e. prestained markers will run differently to unstained etc
5. you have 2 forms of your protein one which is detected by WB (this is the larger fragment and may contain the epitope site for Ab) but is in very low amounts and cant be seen by staining.
If in doubt run samples on a SDS-PAGE with all the relevant markers on each side. Cut gel in half. WB one half and stain the other.
You have to check for contaminants and your prepared gel I go with Jake Hermanson answer
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