Free Thiol Assay Kit (Fluorometric) (ab112158)

1. The kit can only quantify thiol groups, not disulfide bonds.

2. DTT cannot be used as reducing agent because it contains -SH groups, and these will interfere with this assay. That is also the case for beta mercaptoethanol. If either has to be used to reduce the disulfide bond of your protein sample, it needs to be fully removed. You may try de-salting spin column to remove the lower molecular weight molecules.

3. It is not unusual to see a relative decrease in protein signal compared to standard signal over time. Although the signal increases with incubation time for the standards and your protein sample, the increasing rates are different. The standard is GSH, a small molecule,. The reaction rate is much slower for large molecules. We recommend reading at 10 to 20 minutes as long as the sample is detectable. If the sample concentration is very low and needs a longer incubation time to be detected, you can select a protein which has a molecular weight and number of -SH groups similar to your sample as standard instead of GSH.

This kit is very sensitive to presence of DTT, beta mercaptoethanol or guanidine HCl. Even nanomolar quantities of these reagents will negatively effect the results. Please ensure your samples are free of these reagents before using this kit.

The Thiol Quantification Assay Kit (Fluorometric) (ab112158) should be compatible with both Urea or Guanidinium-HCl and should not interfere with the assay.

The kit is provided with glutathione (GSH) as a standard. This molecule contains one cysteine molecule and could therefore be used to calibrate the assay.

Thank you for your email and your interest in our products.

I can confirm that regrettably TCEP and all other reducing agents such as DTTor b-mercaptoethanol will interfere with the Thiol Quantification Assay Kit (Fluorometric) (ab112158).

I hope this answer is of help nevertheless. Please do not hesitate to contact me again if you require further information.

Thank you for contacting us.

I have heard back from the lab with the following advice:

A solid black microplate (black walls and black bottom) would bepreferably recommendedover a black-walled microplate with this assay.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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I can confirm that using bacterial samples should be ok as long as the bacteria is lysed. The detection limit of the kit is 10 nM, so as long as the there is 10 nM cysteine differences among the mutations, it should be OK. Please note this has not been specifically tested on bacterial samples.

Thank you for contacting us.

Yes, the kit can be used for endpoint read. I recommend you read at 15min, 30 min and 1 hour see which one is best for your experiment. The Ex/Em=490/520 nm.


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Please see my math below calculating the minimum volume of your sample needed if we assume the final molar concentration of free cysteine is 25% of the molar concentration of cysteine originally in your sample (50% from 1:1 reduction in DTT or TSEP, then 25% due to reducing expected to release 50% of disulfide bonds, releasing the cysteine). This also assumes that the 14kDa molecular mass of your molecule is directly proportional to the molecular mass of cysteine in your sample since there is 1 cysteine group per sample molecule.



Molar mass of cysteine = 121.16 g/mol, this is 1/115.55 the molar mass of your molecule



14 kD = 14,000 g/mol (1mL/.00075g) = 1.8666 X 10^7 mL/mol (1L/1,000mL) = 1.8666 x 10^4 L/mol = 5.36 x 10^-5 M ***starting molar concentration of your molecule



5.36 x 10^-5 M (1 molar mass unit of your molecule / 115.55 molar mass units of cysteine) = 4.64 x 10^-7 M *** starting molar concentration of cysteine



0.25 x 4.64 x 10^-7M = 1.16 x 10^-7 M



The kit is capable of detecting 1 picomole of cyseine in a 100 uL assay volume, which is 10 nM.



c1V1 = c2V2

1.16 x 10^-7 M (x) = 10 x 10^-9 M (100 uL)

x = 8.62 uL



So theoretically if all of the assumptions and calculations above hold true, you could use a minimum of 8.62 uL of your post-reduction sample per well, although I would recommend using more than this if possible.

Theoretically TCEP should not effect this assay but it does react with the dye. TCEP seems to react with a lot of the things but unfortunately we are not sure how this occurs.