Dear All,
I just have read the following statement, that to maintain the biological activity of peptides after RP HPLC elution.
"after eluting fractions, the protein(s) or peptides of interest should be collected directly into buffer with subsequent removal of organic solvents before lyophilization".
can any one give more explanation for it.
thank you.
Hi Mohammed,
Biological activity is usually requires an intact protein/peptide (higher order) structure. Since the organic solvents used to elute proteins/peptides from a reversed phase C18 column can disrupt protein/peptide structures, it is recommended to evaporate any organic solvent after RP elution (and reconstitution in a suitable physiological buffer) to minimize denaturation.
Hope this helps!
I agree with the previous answer, but you have to make sure that the denaturation due to organic solvents is reversible after solvent evaporation ...
Thank you to Eef Dirksen & Arnaud Delobel for your valuable answers, but i have two issues: first one, Eef Dirksen reconstitute it in physiological buffer do you suggest phosphate buffer saline at 7.4 pH a general buffer used, but even after that i have to lyophilize it again.
Dear Mohammad, i think effect of Acetonitrile/Water as an eleunt has less effect on Biological activity of purified peptides compared to other hydrophilic solvents like methanol. I agree with Eef Dirksen to go for reconstitution in a suitable physiological buffer (or the buffer which you have to use for Assays be it for animal cells or microbial cells) to minimize denaturation even if you have to go for Lyophilization again. The idea behind the reconstitution and then lyophilization is to maintain constant ionization state of different functional groups in peptides, in other words you can call it "pH memory".
thank you so much Dr YOUNIS RATHER, I sent to you a personal message please have a look on it.
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