I am looking to estimate the free thiols in my Polymer using the NTSB assay ( Thannhauser et. al. , Methods in Enzymology, 143 , 115 – 119) . In this assay the chromophoric agent is NTB which produced after reacting with free thiol in the analyate. The ratio of NTB to analyate is 1:1 , and hence measuring NTB concentration amounts to measuring the analyte's concentration.
I have tried to measure the concentration of a known sample (Beta mercaptoethanol and glutathione) using the molar extinction coefficient (E) mentioned in the literature. Unfortunately, the E value mentioned varies from paper to paper(13900 Mcm-1 or 13600 Mcm-1). As a result of that, the concentration obtained from spectroscopy doesn't match the actual concentration. I have thought of making a standard curve and comparing the value of the unknown analyate to that standard curve. Since the concentration values of BME and glutathione do not match the expected values, they are off the candidate list. Do you have suggestions on a free thiol (-SH) standard?
As I have seen, the extinction coefficient is 14.15 mM -1 cm-1 at 412 nm.
But you can use a cysteine solution (as free thiol standard) for the calibration curve (the OD values should be lower than 1.8). Your result can be expressed as micromols of cysteine.
Thank you for your reply. Could you please share the citation for the extinction coefficient ??
L Cysteine is a good thiol standard for quantitative analysis of thiols
No, it is not necessary.
See the atachement, Cys on the bottom of page, on the graph.
I have tried using L Cystine * HCl and Cysteamine HCl despite that also the number are off by a major margin. I have tried changing the incubation time. Initially, I placed the sample in dark for 25 mins, and in the second trial for 5 mins.
I have a feeling the problem is with the instrument. Do you have suggestions on spectrometer standards ?
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