I have a project studying the activity of an enzyme in vitro. My assay for activity is run in 5 mM MOPS buffer pH 7.2 and I can't change this. Optimally, after purifying my enzyme from an E. coli prep I would store it in a similar buffer with some glycerol and DTT. My first question is does anyone know of a reason why MOPS shouldn't be used for long-term storage (eg compared to phosphate buffers)? Second, could I get away with a low MOPS concentration (closer to 5 mM than 50 mM)? Third, do I also need to have salt (eg for stability or solubility) and at what concentration?
Thanks for any insights!
Hi
In general buffers may precipitate or concentrate during freezing leading to pH fluctuation but if you add 20-30% glycerol then they shouldn't create problems, addition of glycerol also useful in stabilizing your protein.
Hope this helps
MOPS in aqueous solution is stable at room temperature for a little less than a year and will develop a slight yellow color as I've experienced from SDS-PAGE work. You will not have any issue at -80C if long-term storage means a few years. However, have you considered the pKa? The pKa of buffers is dependent upon temperature and therefore the final pH is affected by the temp. See what happens to Tris. MOPS's pKa is fairly constant over the various temperatures used by investigators while working with their buffers but I don't know what it is at -80C, but it will be stable. I've gone as low as a few mM in buffer concentration as long as the pH is in the middle of the buffering range and pH7.2 is the case for MOPS. So then low conductivity could be come an issue depending upon the protein concentration but you will not likely need to go to physiological NaCl concentration, especially in the presence of glycerol which lowers the effective water concentration and thereby compensates for loss of conductivity due to low temperatures and low salt concentrations. Keeping reduced DTT present is critical as well as preventing phase transitions so having glycerol present is very important.
5 mM is a low concentration for a buffer, meaning it takes very little acid or base to change the pH. I would store the protein in a concentrated form in 50 mM buffer. You can then dilute it into the reaction with the lower buffer concentration. MOPS should be OK for storage at -80. As for the salt, that depends on the enzyme. Some enzymes like salt and some don't. You can find out about yours by testing the effect of salt on its activity. I think it is a good idea to include 20% glycerol in the storage buffer as a stabilizer, and also because it helps the sample to thaw out more quickly.
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