We are working with a reductase enzyme that is very unstable at dilute concentrations (functional dimer, likely dissociating when dilute). We've explored increasing salt and buffer concentrations,, varying buffers, salts, and pH, and increasing glycerol concentration. The addition of BSA to the assay conditions seems to greatly help, but we are losing activity with our control inhibitors, likely due to protein binding (very lipophilic compounds). I'd like to know if there are other innocuous proteins that we can try other than albumin? Thanks in advance for any suggestions from the community. KH
I cannot be sure that these will help in your assay - but, bovine gamma globulin or mouse IgG might be fine for your purposes. Also, rabbit muscle aldolase.
https://www.thermofisher.com/order/catalog/product/23212
https://www.sigmaaldrich.com/catalog/product/sigma/i5381?lang=en®ion=US
https://www.sigmaaldrich.com/catalog/product/sigma/a8811?lang=en®ion=US
I would tend to try these when BSA or other "protective" reagents don't cut it (or, more, when BSA IS interfering for whatever reason) because we have them at hand and they are usually non-interfering.
Another possibility is to spike in (1x or) 3xFLAG peptide - this has worked well for me as a "blocking" agent, preventing the loss of my target complexes on plastic surfaces etc. and thus preserving their activity.
It is quite hydrophilic I believe.. this may be of use for you given your comment on inhibitors and lipophilicity.
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