I have dimeric and oligomeric forms of a 4,26 KDa peptide, it has positive net charge and is hydrophobic. I need to test the monomer in the antimicrobial assay.
The problem is that even if you isolate 100% monomer, what will stop it from forming dimers and higher order oligomers either before or during the antimicrobial assay?
Agreed, even if you isolate the monomer peak using gel filtration, if you load this peak back onto the gel filtration column you will probably find that it still splits into monomer/dimer and oligomer peaks.
However, you could try altering the buffer (increase or decrease salt/pH) or concentration to see if this improves matters.
Before, I used to use a finally step with HPLC or Sep-pak and then always got monomeric form also with activity. Now I have tried to avoid any contact with solvent and that is why I changed the protocol. Now also I have much more mass of protein than before. It shall be that the conditions in which the samples dried after AcN and TFA, it was desirable to prevent aggregation? After dried, I allways use water for reconstitution.
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