I am expressing two bacterial proteins in E.coli
one is truncating and appearing 7 KDa below the actual size on sds.
another protein is forming oligomer or dimer and giving extra band at higher molecular weight along with normal protein, i am visualizing two bands of proteins expressing at different size on sds. suggest me the future trouble shooting..
thanks in advance.
Dear Naresh Kumar Manda
For avoiding the truncation - use protease inhibitors at first stages of purification and purify at 4 C, if possible.
In the case of dimers/oligomers - add fresh reducing agent (DTT, 2-mercaptoethanol, etc.) and incubate at 95 C for 5 minutes before the electrophoresis.
To avoid eventual proteolysis you would use PMSF at 0.5 or 1mM final.
If you are not sure that the product 7kDa lower than expected, you can perform a Western blot using an antibody against to your protein or the purification tag (anti Histidine for example).
Regards
Noe
Deatr Naresh Kumar Manda
the truncated forms are present in the total and soluble expression before purification or only after purification?
At what temperature did you performed protein expression? 37°C or lower?
There are many possible reason why a protein is degraded in E:coli expression and it depends from the intrinsic protein sequence, so is not so simple to provide you details.
On the other hand is quite strange that in denaturing SDS-page with reducing agent do you see something bigger (oligomer) since theorerically in this condition you see in the gels only the monomers and not oligomers.
An expection may happen if you have in the gel hydrofobic regions (eg for memmbrane proteins) or too many cisteines and your reducing agent is not enough to reduce all.
Ciao
Manuele
Prof manuel marnelli
I am expressing at 37 degrees.
After Ni-NTA purification i am seeing that my protein is getting truncated and appering approx 7 KDa below the actual size.
Hi,
First, you need to determine whether your target protein is expressed, observe the peak position of the gel filtration chromatography, or let the SDS-PAGE gel be subjected to mass spectrometry analysis.
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