It appears that some users have encountered an error code while troubleshooting a dtnb test. This problem can occur for several reasons. Let’s discuss this now.

DTNB reacts with a free sulfhydryl group to form a disulfide and 2-nitro-5-thiobenzoic acid (TNB). The target of DTNB in ​​this response is the conjugate base (RS-) of the free sulfhydryl group.

Thermo Scientific Pierce Ellman’s Reagentagent (DTNB) reacts with sulfhydryl groups to form a colored product and is a proven method for measuring reduced cysteines as well as other free sulfhydryls in solution.

How do you dissolve in DTNB?

DTNB (0.1 M) can be buffered or suspended directly in distilled water (no buffer) and slowly titrated with 1 M Tris base at pH 7.5 avoiding pH 9 to prevent hydrolysis preventing disulfide degradation. Solutions can be stored frozen for countless weeks.

Ellman’s reagent (5,5′-dithio-bis-[2-nitrobenzoic acid]) is used to approximate the sulfhydryl groups in the sample, as well as to compare with a standard sulfhydryl compound such as cysteine. Alternatively, sulfhydryl groups can be tested using the TNB extinction coefficient (14,150 M -1 cm -1 at 412 nm). Ellman’s reagent was used not only for the determination of sulfhydryls, for example, for the determination of alkylthiols using HPLC using the pre-column derivatization method, but also for the search for thiols in the active sites of some enzymes.

• Sulfhydryl assay – reacts quantitatively with free (reduced) sulfhydryl groups (-SH) to give detectable TNB product

Color detection

– Colored equipment allows spectrophotometric measurements in cuvettes or possibly on a microplatethose (λmax = 412 nm; μ means 14 150/M cm)

Proven Method

– A well-characterized chemical analysis allows one to quantify the extinction coefficient of the sulfhydryl group of a peptide or protein, calculate it or check it against a standard.


The cysteine ​​DTNB reacts through the free sulfhydryl group, exposing the sugar-formed mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB). The target of DTNB during this reaction is the conjugate root (r-s-) of the free sulfhydryl number. Therefore, the rate of this reaction depends on several factors: (1) the actual pH of the reaction, (2) this pKa’ sulfhydryl, and (3) steric effects in addition to electrostatic effects. TNB is a decorated substance formed in this reaction and has a high molar termination factor in the visible range. Ellman (1959) first reported the molar extinction coefficient of TNB on the way to 13,600 M-1 cm-1 at Nm 412 and pH 8.0. Therefore, these advantages are often mentioned in modern literature. However, subsequent studies have shown thatthen the molar extinction coefficient is more accurately represented by the value level of 14,150 M-1 cm-1 at 412 nm. TNB absorption may not be affected by changes in pH from 7.6 to 8.6. However, the quench-related TNB varies in different solvents.

My protein is buffered in Tris pH 7.5 and contains free-looking cysteine. I tried twice, but one person had to measure the absorbance at 412 nm.

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Francis Laboratories, c/o SLT Dept, Rivers Polytechnic Institute

I think Adam is right. My husband and I prefer to use a buffer for this.

How do I create a DTNB reagent?

Make a stock solution of 10 mM DTNB by dissolving 40 mg DTNB in ​​10 ml DMSO. The stock solution is stable for 3 months at 4°C. Dilute the stock formulation 100 times with 0.1 M Tris-HCl, pH 7.5 to make a 0.1 mM DTNB stock solution.

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dtnb assay troubleshooting

Try building a standard curve using DTT as a source of thiols (note that DTT contains thiols/molecules) 2 This should allow you know if the system is working properly.

Oak Ridge National is Labor

Here is a protocol you might try:


Reaction buffer: 0.1 M phosphate buffer, pH 8.0.

Denaturation buffer: 6 M guanidinium nyl chloride, 0.1 M Na2HPO4, pH 8.0.

(It is strongly not recommended to use urea inside or in place of guanidinium hydrochloride as it easily breaks down into cyanates which can react with thiol groups)

Elman’s solution: 10 mM (4 mg/ml) DTNB in ​​0.1 M phosphate buffer, pH 8.0.

If a new one is not purchased, it is recommended to recrystallize DTNB from aqueous ethanol

Dithiothreitol (DTT) solution: 400 mM in distilled water Clothes

May’s Free Thiol Analysis

It is necessary to introduce thiol groups that can be captured inside the desired protein. Therefore, the sample can be mixed in a buffer reaction or to prevent denaturation. A known highlighting solution should be prepared with a protein-free research mixture. Sufficient protein should be used so that, in many cases, typically one thiol per protein molecule can be detected; in practice, at least 2 nmol of protein (in 250 µl) is usually required.

A sample and plan containing 3 ml of Reaction Shield or denaturation buffer should be prepared and read at approximately 412 nm. The absorbance must be properly adjusted to zero (A_buffer).

dtnb assay troubleshooting

Add 100 µl screen to the reference sample.

Add 100 µl of Ellman’s solution to dilute the sample. Write absorbance (A_DTNB).

How do you dissolve Ellman’s Reagent?

Dissolve 4 mg/mL of Ellman’s reagent, 5,5′-dithio-bis-(2-nitrobenzoic) acid (DTNB) in reaction buffer (Ellman’s reagent solution). Dissolve 5.268 mg of cysteine ​​hydrochloride monohydrate (MW = 175.6) in reaction buffer to an initial concentration of 1.5 mM (standard A in the dining table below).

Add 100 µl of dissolved protein for comparison.

Finally, use 100 µl of the sample-specific protein solution and, after thorough mixing, remove the absorbance until no further increase becomest possible. This may take several minutes. Pay attention to the remaining value (A_final). Concentration

The amount of thiols present can be further calculated from molar absorbance including the TNB anion.

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Alexander gave some useful tips.

If your control turns yellow (which means DTNB is working) and you are consuming enough protein (how do you determine the concentration?), then most likely you are not lowering your protein level. This may be automatically associated with restorative, insufficient obstruction, or latent disulfides (or both). Quite often, the failure to respond to all DTNB-related obstacles is due to a reduced protein content.

For reconstitution, we typically used a freshly prepared solution of 10 mM DTT in 20 mM Tris-degassed 1 mM HCl, EDTA buffer, pH 8.0. EDTA and degassing are generally important to inactivate divalent ions and minimizeretention of dissolved oxygen. Do not run your TNT solution – it is fresh just before use. For SH you should include a total of 6M guanidinium hydrochloride or 8-10M urea. After incubation for 1 hour at room temperature, pass the cause through a PD-10 or NAP-5 desalting column previously equilibrated with a degassed obstruction (20 mM Tris-HCl, 1 mM EDTA, pH buffer, 8.0). They will want to help you work as quickly as possible and avoid air oxidation. We usually work in a glovebox with surrounding inert materials. Your newly recovered protein will be within the fractions expected by the column manufacturer. Be careful not to contaminate the cavity containing your protein with the volume containing DTT.

Dtnb 분석 문제 해결
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Fehlerbehebung Bei Dtnb Assays
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