Good morning.
I have two mutants of the same protein that differs one from the other by only one aminoacid (Leu/Ala). Unfortunately the samples where partially mixed and I have now a mixture of this two purified proteins.
Is there a method that is able to separate such similar proteins? Or I hvae just to start over?
Thank you! Have a nice day!
Hi there,
Such a little difference (exchanging an hydrophobic residue for an other hydrophobic residue) is making both proteins very very similar so you'd better redo the experiments from the start.
Hi there,
Such a little difference (exchanging an hydrophobic residue for an other hydrophobic residue) is making both proteins very very similar so you'd better redo the experiments from the start.
Chromatographic techniques like Liquid chromatography & Gel Filtration Chromatography will solve your issue.Liquid chromatography used to separate mixtures of proteins.
proteins that differ in mass can be separated by gel filtration chromatography.By use of proteins of known mass, the elution volume can be used to estimate the mass of a protein in a mixture.
How big are your proteins? Depending on how much work you want to put in it *might* be possible to separate them into organic, and aqueous layers given the right pH, if you calculate their overall charges. It might also be possible to try HPLC on a slow run to separate peaks.
I've never worked with that exact problem, but getting creative with salting out, electrophoresis, or chromatography can help. Unfortunately it's usually easier, cheaper, and more efficient to begin again.
Dear Silvia,
I suggest Reverse Phase HPLC (RP-HPLC) to separate the proteins. You can set a slow flow rate at 0.3 mL/min by Acetonitrile. Depend on the size of the proteins, you can use C4 or C8 column.
If you decided to separate them, I can suggestion a protocol.
Good luck
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