x*******i
发帖数: 32 | 1 Hello There,
We have a protein (pI = 8.9, 10 mg/mL, MW 100 K) precipitated in 10 mM
phosphate buffer (pH around 7) after overnight storage at 4 degree. The
precipitate will be soluble again when warming up solution to room
temperature. Addition of NaCl will prevent precipitation. Other common
buffers are OK not causing precipitate.
Hypothesis is ionic cross-linking. Phosphate ion has intense negative charge
density and working as the cross-linking agent, its multiple negative
charges interact with positive proteins to cause precipitation.
The ionic interaction between negatively charged phosphate and positively
charged protein is supposed to be weak and transient. Perhaps only a small
percentage of phosphate ions in buffer are enough to initiate precipitation.
Is there any direct way to prove the above hypothesis?
Thank you very much! | s*******e
发帖数: 1010 | 2 interesting question although it may not lead to Nature paper.
before u propose protein-ion interaction, I think u should make sure the
precipitation is ur protein and it is phosphate-dependent.
use HEPES as buffer and add series conc. of Pi to test dose-response. Only
difficulty is to quantify protein precipitation, but u could spin down it
and determine protein conc in supernatant. |
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