When and why would you quantify the number of free thiols in a sample?
Quantifying the number of free thiols in a sample can be useful in a number of applications. For example, if the final purpose is the conjugation of a thiolated molecule to a maleimide – activated label, quantifying the number of free thiols on the molecule is highly recommended in order to have a more controlled conjugation reaction.
What is the minimum thiol concentration detected with the assay?
A concentration of 1.5 µM of thiols can be easily detected.
How do I remove the thiolating agent before testing?
Dialysis of thiolated antibodies/ proteins is not recommended due to the consequent big dilution. The thiolating reagent can be removed using a small size-exclusion/gel filtration gravity or spin column. Consult manufacturers’ instructions for use and select resin for your needs.
My buffer is outside the recommended ranges, what should I do?
If the buffer is too concentrated, dilute the sample to within buffer range. If the sample cannot be diluted further, the buffer could alternatively be exchanged using a size exclusion/gel filtration gravity or spin column.
What if the measured absorbance of the sample is out of the range set by the calibration curve?
Concentrate or dilute the sample in order to be within the linear range of the calibration curve.