The enzyme is a functional dimer that is losing activity rapidly when diluted to low concentrations (e.g. 50 nM). We believe this is due to dissociation. We have optimized the buffer and pH (MES, pH 6.5). Considering higher salt concentration (how high can we reasonably go?) and addition of BSA to assay buffer (what concentration?). Any tips or tales of experience with this issue would be most appreciated.
You can avoid having to dilute the protein so much if you use a shorter reaction time.
I don't think adding BSA will prevent dimer dissociation since it will not affect the Kd. Changing the salt concentration, pH, and temperature could have an effect. There could also be an effect of specific ions, of substrates, or of allosteric regulators.
BSA could be beneficial by acting as a blocking agent to prevent the protein sticking to surfaces. A low concentration of a nonionic detergent (e.g. Tween-20, Triton X-100, Brij-35) can have the same benefit.
Adding a high concentration of a sugar (e.g. 1 M sucrose, 0.5M trehalose) can have a stabilizing effect.
Dear Kirk:
BSA and glycerol (0.5-20 % w/v) work fine in some cases promoting structure stabilisation. Is your protein and halophilic protein? If so, try to increase salt concentration in your buffer (NaCl or KCl up to 4 M).
Good luck!
You could consider crosslinking to prevent disassociation. Of course, the chemicals cause the crosslinking may deactivate your protein,
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