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Free Thiol Assay Kit (Fluorometric) (ab112158)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (1)Q&A (16)References (2)

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Functional Studies - Thiol Quantification Assay kit (Fluorometric) (ab112158)
  • Functional Studies - Thiol Quantification Assay kit (Fluorometric) (ab112158)
  • Functional Studies - Thiol Quantification Assay kit (Fluorometric) (ab112158)
  • Functional Studies - Thiol Quantification Assay Kit  (Fluorometric) (ab112158)

Key features and details

  • Assay type: Quantitative
  • Detection method: Fluorescent
  • Platform: Microplate reader
  • Assay time: 20 min
  • Sample type: Cell culture extracts, Plasma, Urine

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Overview

  • Product name

    Free Thiol Assay Kit (Fluorometric)
    See all Thiol kits
  • Detection method

    Fluorescent
  • Sample type

    Urine, Plasma, Cell culture extracts
  • Assay type

    Quantitative
  • Assay time

    0h 20m
  • Product overview

    This product is not suitable for protein samples.


    Free Thiol Assay Kit (Fluorometric) (ab112158) provides an ultrasensitive fluorometric assay to quantitate thiol content that exists in small molecules such as free cysteine, glutathione, and cysteine residues. The assay is based on a proprietary non-fluorescent dye tha generates a strongly fluorescent adduct upon reacting with a thiol compound. The signal produced by the adduct can ebe asily read by a fluorescence microplate reader at Ex/Em = 490/520 nm. In addition, both absorption and emission spectra of the thiol adduct are pH-independent, making the assay highly robust. There are few reagents or assay kits available for quantitating thiol content in biological systems, and the majority of commercial kits either lack sensitivity or have tedious protocols. The assay can detect as little as 1 picomole of cysteine or GSH in a 100 µL assay volume (10 nM). 


    Please note it is not possible to do a quantification in comparison to the standard.


    Free Thiol assay protocol summary:
    - add samples and standards to wells
    - add reaction mix
    - analyze with microplate reader for 10 min to 1 hr

  • Notes

    This product was previously called Thiol Quantification Assay Kit (Fluorometric).

    This product detects thiol groups on small molecules. If you want to detect thiol groups in proteins, we recommend Total Protein Thiol Quantification Assay Kit (Colorimetric) (ab219272).

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 200 tests
    Assay Buffer 1 x 25ml
    DMSO 1 x 200µl
    GSH Standard 1 x 62µg
    Thiol Green Indicator 1 vial
  • Research areas

    • Cell Biology
    • Other Antibodies
    • Drugs and Chemicals
    • Kits/ Lysates/ Other
    • Kits
    • Cell Metabolism Kits
    • Other Metabolism Assay
    • Kits/ Lysates/ Other
    • Kits
    • Molecular Biology Kits
    • Protein Quantitation Kits
    • other

Associated products

    Images

    • Functional Studies - Thiol Quantification Assay kit (Fluorometric) (ab112158)
      Functional Studies - Thiol Quantification Assay kit (Fluorometric) (ab112158)

      Standard curve: mean of duplicates (+/- SD) with background reads subtracted

    • Functional Studies - Thiol Quantification Assay kit (Fluorometric) (ab112158)
      Functional Studies - Thiol Quantification Assay kit (Fluorometric) (ab112158)

      GSH measured in cell lysates showing quantity (nmol) per 1 mln cells.

      Samples with the concentration of 1e8 cells/mL were used. Samples were diluted 100-1000 fold.

    • Functional Studies - Thiol Quantification Assay kit (Fluorometric) (ab112158)
      Functional Studies - Thiol Quantification Assay kit (Fluorometric) (ab112158)

      GSH measured in biological fluids. Samples were diluted 10-1000 fold.

    • Functional Studies - Thiol Quantification Assay Kit  (Fluorometric) (ab112158)
      Functional Studies - Thiol Quantification Assay Kit (Fluorometric) (ab112158)
      GSH and Cysteine dose responses were measured in a 96-well black plate with ab112158 using a microplate reader. As low as 10 nM (1 pmol/well) of GSH or Cysteine can be detected with 10 minutes incubation time (n=3). The insert shows the low levels of thiol detection.

    Protocols

    • Protocol Booklet

    Click here to view the general protocols

    Datasheets and documents

    • Datasheet
    • SDS
  • References (2)

    Publishing research using ab112158? Please let us know so that we can cite the reference in this datasheet.

    ab112158 has been referenced in 2 publications.

    • Seo Y  et al. Assembly and Folding Properties of Cytosolic IgG Intrabodies. Sci Rep 10:2140 (2020). PubMed: 32034177
    • Marino-Puertas L  et al. Recombinant production of human a2-macroglobulin variants and interaction studies with recombinant G-related a2-macroglobulin binding protein and latent transforming growth factor-ß2. Sci Rep 9:9186 (2019). PubMed: 31235767

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-10 of 17 Abreviews or Q&A

    Question

    I am performing the Thiol Quantification Assay (ab112158) on intact proteins and I was wondering if the assay reads disulfide bonds, or just thiol groups? In other words, will I be able to quantify the degree of reduction of my samples? Also what reducing agent is suggested if DTT may yield inaccurate quantification? Would repeated buffer exchange suffice? Also, I performed the assay where I read the same plate at different incubation intervals. The concentration of my samples against the standard curve for each time point decreased as the incubation time increased. (10 minute incubation resulted in a higher calculated concentration in my samples than 60min incubation). What could be causing this change, and what is the recommended incubation for protein samples?

    Read More

    Abcam community

    Verified customer

    Asked on Nov 25 2013

    Answer

    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.

    Read More

    Tom Ruyle

    Abcam Scientific Support

    Answered on Nov 25 2013

    Question

    With what levels of DTT, beta mercaptoethanol and guanidine HCl is this kit compatible?

    Read More

    Abcam community

    Verified customer

    Asked on Aug 09 2013

    Answer

    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.

    Read More

    Jeremy Kasanov

    Abcam Scientific Support

    Answered on Aug 09 2013

    Question

    Is it possible to use denatured protein within the assay? Do denaturants such as Urea or Guanidinium-HCl interfere with the assay?

    Do you know a suitable protein positive control for this assay (for absolute thiol quantification) with maybe only one cysteine?

    Read More

    Abcam community

    Verified customer

    Asked on Apr 30 2013

    Answer


    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.

    Read More

    Abcam Scientific Support

    Answered on Apr 30 2013

    Question

    I wondered if it is possible to use TCEP within the assay. Does TCEP or other reducing agents interfere with the dye of the assay? Does TCEP have to be removed from a protein sample before measuring?

    Read More

    Abcam community

    Verified customer

    Asked on Feb 25 2013

    Answer

    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.

    Read More

    Abcam Scientific Support

    Answered on Feb 25 2013

    Question

    should a solid black microplate (black walls and black bottom) be used or rather a black-walled microplate?

    Read More

    Abcam community

    Verified customer

    Asked on Jun 18 2012

    Answer

    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.

    Use our products? Submit an Abreview. Earn rewards!
    https://www.abcam.com/abreviews

    Read More

    Abcam Scientific Support

    Answered on Jun 18 2012

    using the kit for thiol quantification after flies lysis

    Good Excellent 5/5 (Ease of Use)
    Abreviews
    Abreviews
    the kit is very easy to use. it contains very low amount of assays though.

    Mrs. Smadar Tabenkin

    Verified customer

    Submitted Sep 03 2014

    Question

    I have a question regarding the Thiol Quantification Assay Kit. The FAQ says that it is suitable for mammalian cells but can it be performed on bacteria? Is it sensitive enough to detect difference in cysteine levels between mutants?

    Regards,

    Read More

    Abcam community

    Verified customer

    Asked on Sep 02 2014

    Answer

    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.

    Read More

    Sam Washer

    Abcam Scientific Support

    Answered on Sep 02 2014

    Question

    Name: Ben Mayes
    Product code: 112158
    Lot number: GR167278-7

    Inquiry: Hello, I recently ran the Thiol Quantification Assay Kit, Cat. # ab112158. Can this kit be read as an endpoint read? We do not have the capability to read the plate kinetically. If it can be read as an endpoint read, can you recommend an appropriate read time and wavelengths? Thanks Ben

    Read More

    Abcam community

    Verified customer

    Asked on Aug 07 2014

    Answer

    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.

    Read More

    Heather Allen

    Abcam Scientific Support

    Answered on Aug 07 2014

    Question

    What is the minimum volume of sample I should add to meet the sensitivity of this kit?
    I have an antibody with 1 cysteine group per molecule that is 14 kD. The starting concentration of my sample is 0.75 mg/ml, which is then diluted 1:1 in DTT or TSEP for reduction (down to 0.375 mg/ml). Reducing is expected to release ˜50% disulfide bonds (0.1875 mg/ml).
    Kit sensitivity is 1 nM cystein.

    Read More

    Abcam community

    Verified customer

    Asked on May 16 2014

    Answer

    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.

    Read More

    Kevin Hanson

    Abcam Scientific Support

    Answered on May 16 2014

    Question

    I see from the Q and A that TCEP is not compatible with this kit. Since TCEP does not have a -SH group I'm just wondering how this compound will interfere with this assay?

    Read More

    Abcam community

    Verified customer

    Asked on Apr 02 2014

    Answer

    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.

    Read More

    Jeremy Kasanov

    Abcam Scientific Support

    Answered on Apr 02 2014

    1-10 of 17 Abreviews or Q&A

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