Can anyone tell me until which pI difference I can seperate two proteins with the IEX? Is a pI difference of 0.5 still possible to seperate?
Hi Timm,
I guess that depends on the absolute pI values of the proteins of interest, the column you're planning to use, whether you're thinking of running a salt gradient (and at which pH) or a pH gradient (or a combination of the two) and whether there are perhaps other physicochemical properties, such as protein size or hydrophobicity that could be used to separate both proteins.
Hope this helps, some more information from your end definitely would :)!
I agree with Eef. Weak AEX or strong AEX has an enormous difference on the pH/salt gradient one would use. If pI is too close for baseline resolution without significant DoE, I would recommend a MMC column such as Capto MMC (wCEX + hydrophobic interactions) or Capto Adhere (sAEX + HIC) where you can use another layer of abstraction to isolate your molecule of interest. Once bound to a MMC column, you can use chaotropes (CaCl, Urea, etc) and arginine to weaken or strengthen particular ionic/hydrophobic interactions with the stationary phase of the column - we call them 'modulators'.
These resins are so good that companies are beginning to move away from ProteinA affinity for isolation of mAbs.
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