8 Comments

Go to the profile of mdfenko
mdfenko 7 months ago

in what medium do you isolate the protein (what are the components and composition)?

if you have dtt or 2-me in the medium, in relatively high concentration, then you may be breaking the disulfide bonds which hold the dimer together.

Go to the profile of pkh28
pkh28 7 months ago

my lysis buffer: 50mM potassium phosphate, PH7.5-150 mM nacl, 5mM mgcl2,25 mM imidazole and 200 micromolar TCEP
elution buffer with 500mM imidazole and SEC buffer all the same without the addition of imidazole

Go to the profile of relaxin
relaxin 7 months ago

Some proteins have a tendency to bind with each other to form dimers or multimers without the involvement of disulfide bonds. If you run your purified protein on gel filtration column with native buffer, your protein may appear as a molecule with MW of dimer. But if you run it on SDS-gel, then it appears as monomer.

Sometimes heterodimer (of two different molecules) is also possible. You can separate them on SDS-gels, ion-exchange chromatography in the presence of urea, or gel filtration in the presence of guanidine HCl.

Go to the profile of pkh28
pkh28 7 months ago

Thank you for your attention, but my problem is that the protein supposed to be in dimer form while it is coming out of gel filtration chromatography as a monomer.
The protein is not active, I was wondering if it might be due to the monomer form??

Go to the profile of Roberto Rosati
Roberto Rosati 7 months ago

Chiming in: when you write,

do you mean that you're running a native PAGE and you see the protein as monomer? Or if not, what other technique are you using? This might help troubleshooting the issue.

Go to the profile of mdfenko
mdfenko 7 months ago

pkh28 is apparently using sec (used to be called gel filtration) on a column.

the tcep is probably separating the dimer. try using a much lower concentration.

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pkh28 7 months ago

I have checked it with Analytical Ultracentrifugation, and it is a monomer. I also tried SDS-page with and without DTT, it is showing the band corresponding to the monomer size.

Go to the profile of relaxin
relaxin 7 months ago

If it appears as monomer on gel filtration column, it is not disulfide linked. Apparently, a cofactor is removed during purification so that the dimer is dissociated into monomer. You may check the assay buffer components of earlier report for this enzyme, and use the buffer (without substrate) to run gel filtration.