We work with a range of nanobodies (13–17 kDa), and while some go through desalting procedures without much trouble, others essentially disappear on the Zeba™ resin, with recovery rates as low as 25%.
Has anyone come across this problem before? And critically, has anyone managed to resolve it?
Some details:
We follow the manufacturer protocol (Thermo Scientific) to the tee, using the maximal volume applicable to the columns we have (130 µL on the 0.5 mL columns). But for some proteins, even at initial concentrations of around 20 µM, we lose two thirds of our protein and are left with a 7 µM sample of the same volume following desalting. At lower concentrations the relative loss is even greater (e.g. 7 µM pre-desalting → 1.8 µM post-desalting).
The only difference I can identify between the proteins that go through fine (20% loss or less) and the ones that do not (over 50% loss) is the isoelectric point, with the former being acidic and the latter being neutral or alkaline (pI > 7). But I am entirely unsure whether this is related or just a random correlation.
Would love to hear from anyone who has experienced anything similar. Thanks!
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