AAT Bioquest

Amplite® Rapid Colorimetric Total Protein Thiol Quantitation Assay Kit

The Amplite® Rapid Colorimetric Thiol Quantitation Assay Principle
The Amplite® Rapid Colorimetric Thiol Quantitation Assay Principle
The Amplite® Rapid Colorimetric Thiol Quantitation Assay Principle
Ordering information
Catalog Number
Unit Size
Add to cart
Additional ordering information
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
ShippingStandard overnight for United States, inquire for international
Request quotation
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Protein thiols are very important to protein structure, protein function and biological system redox environment. For example, albumin is the most abundant protein in plasma and the free thiol present in the albumin protein are considered as major plasma antioxidants in the body. The change of thiol status in albumin is related to a lot of diseases and disorders, such as kidney disease and Parkinson's disease. Although there are a few reagents or assay kits available for quantitating the total thiol content in biological systems, a key challenge is to have a rapid and accurate method to quantify the amount of free thiol group in a specific protein. Amplite® Rapid Colorimetric Thiol Quantitation Kit provides an accurate method to quantify free thiol group using our proprietary thiol sensor, Thiol Blue™, which has the maximum absorbance at ~680 nm. Thiol Blue™ reacts with the protein samples that contain free thiol groups. The resulted thiol adduct is run through a single spin column to remove the excess Thiol Blue™ sensor, and the absorption spectrum of the purified product is measured. The amount of thiol to protein ratio is calculated from the absorbance ratio of 680 nm and 280 nm. This Amplite® Rapid Colorimetric Thiol Quantitation Kit can be performed in a traditional cuvette, NanoDrop™ Spectrophotometer or a convenient 96-well absorbance plate reader with a UV-transparent plate.



Absorbance250 nm~750 nm
Recommended plateCuvette

Absorbance microplate reader

Absorbance280 and 680 nm
Recommended plateClear bottom


Example protocol


Important notes:
When stored properly, the kit components should be stable for six months. Do not freeze Spin Column (Component C). Warm all the components before run the required assays. 50 to 100 µg of protein sample is needed for determining the amount of thiol amount.


Prepare Sample Solution:

  1. Adjust the volume of 50 to 100 µg of protein sample to 100 µL with Assay Buffer (Component B). Note: The protein sample should be in pH = 6.0 buffer and without DTT or other reagent containing free thiols.

Run Thiol Assay:

  1. Add the protein sample to one vial of Thiol Blue™ (Component A).

  2. Mix them well by repeatedly pipetting for a few times or vortex the vial for a few seconds.

  3. Keep the reaction mixture at room temperature and rotate or shake for 30 - 60 minutes.

Prepare Spin Column for Sample Purification:

  1. Invert the Spin Column (Component C) several times to resuspend the settled gel and remove any bubbles.

  2. Snap off the tip and place the column in the Washing Tube (2 mL, Component D). Remove the cap to allow the excess packing buffer to drain by gravity to the top of the gel bed. If column does not begin to flow, push cap back into column and remove it again to start the flow. Discard the drained buffer, and then place the column back into the Washing Tube. However, centrifuge immediately if the column is placed into a 12 x 75 mm test tube (not provided).

  3. Centrifuge for 1 min in a swinging bucket centrifuge at 1,000x g to remove the packing buffer. Discard the buffer.

  4. Apply 1 mL Assay Buffer (Component B) to the column, let the buffer drain out by gravity, or centrifuge the column for 1 min to remove the buffer. Discard the buffer from the collection tube. Repeat this process for 3 - 4 times.

  5. Centrifuge for 2 minutes in a swinging bucket centrifuge at 1,000x g to remove the reaction buffer. Discard the buffer. Note: Spin Column (Component C) can fit into 2 mL microcentrifuge tubes or 12 x 75 mm test tubes for sample collection during centrifugation. Use the 2 mL microtubes provided with the columns for the initial column equilibration step. Swinging bucket centrifuges capable of generating a minimum force of 1,000x g are suitable for Bio-Spin column use. The gravitational force created at a particular revolution speed is a function of the radius of the microcentrifuge rotor. Consult the swinging bucket centrifuge instruction manual for the information about conversion from revolutions per minute (RPM) to centrifugal or g-force. Alternatively, use the equation to calculate the speed in RPM required to reach the gravitational force of 1,000x g.
    RCF(g) = (1.12 x 10-5) X (RPM)2 X r
  6. RCF = the relative centrifugal force, RPM = the speed of the rotor, r = the radius in centimeters measured from the center of the rotor to the middle of the Bio-Spin column

Purify Reaction Product:

  1. Place the column in a clean Collecting Tube (1.5 mL, Component E). Carefully load the sample (100 µL) directly to the center of the column.

  2. After loading the sample, add 10 µL Assay Buffer (Component B) to the top and centrifuge the column for 5 min at 1,000x g, and collect the solution into the collecting tube.

Run Absorption Spectra with 0.2 mL or 0.5 mL Quartz Cuvette:

  1. Dilute the reaction product by 5-folds with Assay Buffer (Component B) depending on the cuvette size used and the absorbance reading. Note: The dilution factor doesn’t affect the final thiol quantitation result.

  2. Measure the absorption spectrum from 250 to 750 nm, or only read the absorbance number at 280 nm and 680 nm.




View all 1 citations: Citation Explorer
Activation of transcription factors in human bronchial epithelial cells exposed to aqueous extracts of mainstream cigarette smoke in vitro
Authors: Sekine, Takashi and Hirata, Tadashi and Mine, Toshiki and Fukano, Yasuo
Journal: Toxicology mechanisms and methods (2016): 22--31


View all 26 references: Citation Explorer
Covalent surface chemistry gradients for presenting bioactive peptides
Authors: Kipper MJ, Kleinman HK, Wang FW.
Journal: Anal Biochem (2007): 175
Dansyl glutathione as a trapping agent for the quantitative estimation and identification of reactive metabolites
Authors: Gan J, Harper TW, Hsueh MM, Qu Q, Humphreys WG.
Journal: Chem Res Toxicol (2005): 896
Gold nanoparticle-based detection of genomic DNA targets on microarrays using a novel optical detection system
Authors: Storhoff JJ, Marla SS, Bao P, Hagenow S, Mehta H, Lucas A, Garimella V, Patno T, Buckingham W, Cork W, Muller UR.
Journal: Biosens Bioelectron (2004): 875
Fluorometric polyethyleneglycol-peptide hybrid substrates for quantitative assay of protein disulfide isomerase
Authors: Christiansen C, St Hilaire PM, Winther JR.
Journal: Anal Biochem (2004): 148
Assay of total homocysteine and other thiols by capillary electrophoresis and laser-induced fluorescence detection. II. Pre-analytical and analytical conditions
Authors: Bayle C, Issac C, Salvayre R, Couderc F, Causse E.
Journal: J Chromatogr A (2002): 255
A continuous fluorescence assay for sulfhydryl oxidase
Authors: Raje S, Glynn NM, Thorpe C.
Journal: Anal Biochem (2002): 266
Plasma total homocysteine and other thiols analyzed by capillary electrophoresis/laser-induced fluorescence detection: comparison with two other methods
Authors: Causse E, Malatray P, Calaf R, Charpiot P, C and ito M, Bayle C, Valdiguie P, Salvayre R, Couderc F.
Journal: Electrophoresis (2000): 2074
Assays for total homocysteine and other thiols by capillary electrophoresis-laser-induced fluorescence detection. I. Preanalytical condition studies
Authors: Causse E, Issac C, Malatray P, Bayle C, Valdiguie P, Salvayre R, Couderc F.
Journal: J Chromatogr A (2000): 173
Measurement of thiols in human plasma using liquid chromatography with precolumn derivatization and fluorescence detection
Authors: Salazar JF, Schorr H, Herrmann W, Herbeth B, Siest G, Leroy P.
Journal: J Chromatogr Sci (1999): 469
Quantitation of homocysteine in human plasma by capillary electrophoresis and laser-induced fluorescence detection
Authors: Causse E, Terrier R, Champagne S, Nertz M, Valdiguie P, Salvayre R, Couderc F.
Journal: J Chromatogr A (1998): 181