Attempting to produce spectra of SPDP (527 Da adduct on solvent exposed lysines) modified transferrin. I am experiencing solubility issues even at the recommended concentration of 10uM with a buffer system of 49% methanol, 49% water, 2% acetic acid (pH ~3-4). Protein ppt's even with 50% methanol (no acetic acid, neutral pH). I am looking for methods for sample prep of whole protein that people have actually tried and worked in their hands. Thank you!
At a cursory glance, I didn't see volatile buffers in the above references.
If it didn't mention it, the 2 most common volatile buffers used in protein chemistry are ammonium bicarbonate and ammonium acetate. They are around neutral pH when made.
More options:
https://www.nestgrp.com/protocols/trng/buffer.shtml
The buffer you described (methanol/acetic acid) seems to be a fixative (i.e., causing protein denaturation), and it is not surprising at all that the protein precipitated.
The best buffer depends on at which step you are in the sample preparation pipeline. However, either TEAB or ammonium bicarbonate at 100 mM would be a good start point.
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