The protein that I am working on is a 16 kD protein. When I am trying to express this protein in E. coli and performing SDS PAGE I am getting a very faint band at 16 kD but at the same time I am getting a band at 32 kD as well.
This protein is reported to form dimer, so I was wondering if that dimer formation occurs during expression or is it just a random band of some other protein.
* There is just one cysteine in the sequence.
Yes it is possible to get dimer in protein purified in bacterial system, but it is always better you confirm the protein present at 32 kDa using western or MALDI TOF. There is a bacterial chaperon that comes across 37Kda size in bacterial purification system
yes it is absolutely possible to get multimerisation of bacterial proteins as well...if you are performing non reducing SDS PAGE then the presence of a reducing agent like DTT or beta ME should render your protein in the monomeric form, if it is dimerising via di-sulphide linkage... if you still want to confirm whether it is your protein that is getting dimerised then go for a western blot !
Dimer formation can also occur during electrophoresis, once the samples enter the concentrating gel and leave the reductive environment of the loading buffer (see e.g. PMID 8238894) which in your case might be compounded by the exposure, upon SDS denaturation, of an otherwise buried Cys residue.
You can try reducing the protein and then carboxymethylating free cysteines with iodoacetamide before analyzing the sample.
You can run Native PAGE to confirm the oligomer (Dimer) state of your protein and compare the molecular weight of monomer with dimer. It will give you the accurate result plus heating at 100C along with BME for 5-10 minutes is enough to convert the dimer to monomer. On contrary, you can run your sample on Akta HPLC system and can confirm the monomer and dimer states easily.
In bacterial system, most of the time there is a band at ~32-35 kDa conforming to some bacterial protein.To confirm your protein of interest, you should perform a western blot.
Yes, Dimer formation is possible inside bacterial cell. The ~32 kDa band cannot be ur dimer protein, to confirm this do Trypsin in-gel digestion for both 16 and 32 kDa bands separately or simply do Western Blot using antibodies either against the protein of ur interest or against the tag.
By the way how are u purifying the protein,, is it affinity chromatography??
Yes, absolutly possible. Even, runninig a SDS Gel, sometimes, you can see a monomeric band, and also dimeric or multimeric bands as well.
To study the oligomeric state of your protein, you can use analitical ultracentrifugation as well.
It certainly can occur but may not routinely. We made several artificial diploids of different electromorphs of E. coli beta-galactosidase (in which dimer and tetramer do not require disulfide bonding) by introducing the second gene on a plasmid into a wild-type E.coli. We only saw one instance of randomly associated (5 electromorph bands in agarose gels ... admittedly unpublished) . So it is clear that some type of compatibility is critical for dimer or in our case tetramer formation.
I would like to counter your question by saying why shouldn't it form a dimer in bacteria if its known to form a dimer in other systems. Also I guess you are running a denaturing and reducing gel, so most likely its some other contaminating protein.
Other scenarios is: You are running a denaturing non reducing gel(without DTT/Beta mercaptoethanol in ur sample buffer)- In this case if the 32kd band appears and then it vanishes when u add reducing agent, it would proove that the dimerisation is via a disulphide linkage
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