Product Description

3,3’,5,5’–Tetramethylbenzidine (TMB) is a chromogenic substrate suitable for use in ELISA procedures, which utilize horseradish peroxidase conjugates. This substrate produces a soluble end product that is blue in color and can be read spectrophotometrically at 370 or 655 nm. The reaction maybe stopped with 2 M H2S04, resulting in a yellow solution that is read at 450 nm.

Each tablet contains 1 mg of TMB substrate.

The product is available in packages of 50 or

100 tablets. Custom packaging and bulk purchase information available upon request.

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.

Preparation Instructions

0.05 M Phosphate-Citrate Buffer – Dissolve one Phosphate-Citrate Buffer Tablet (Product No. P 4809) in 100 ml of deionized water with stirring to yield a 0.05 M phosphate-citrate buffer, pH 5.0.

OR

Add 25.7 ml of 0.2 M dibasic sodium phosphate (Product No. S 0876), 24.3 ml of 0.1 M citric acid (Product No. C 7129), and 50 ml deionized water. Adjust pH to 5.0, if necessary.

TMB Substrate Solution – Dissolve one 3,3’,5,5’–tetra- methylbenzidine tablet in 1 ml of DMSO and add to 9 ml of 0.05 M Phosphate-Citrate Buffer, pH 5.0. Add 2 μl of fresh 30% hydrogen peroxide (Product No. H 1009) per 10 ml of substrate buffer solution, immediately prior to use.

OR

Dissolve one 3,3’,5,5’–tetramethylbenzidine tablet in 1 ml of DMSO and add to 9 ml of 0.05 M phosphate- citrate buffer, pH 5.0, containing 0.03% sodium perborate (capsules, Product No. P 4922).

Stop Solution - Reaction may be stopped by the addition of 50 μl of 2 M H2SO4 per 200 μl of reaction mixture.

Storage/Stability

Store the TMB tablets at 2–8 °C. Protect from heat, light, and moisture. Allow tablets to reach room temperature prior to use.

References

Bos, E., et al., J. Immunoassay, 2, 187 (1981).

PCS,MAM 01/05-1

Hi Emmanuel,

are you the first to do ELISAA in your lab, or do you have certain problems with your ELISAs?

More details would be really good to give you some fruitful advice!!!

Best,

Kai

Hi Kai,

Usually I use ready-to-use ELISA kits where all buffers were already prepared. This time I have a kit for transferrin quantification where I have only Capture antibody, conjugate HRP and the protein calibrator. I have follow the manufacturer's recommendation and I have had nice results. But I 'am now out of Enzyme substrate solution (because i have taken a old solution of stabilized chromogen from invitrogen).

To build the Enzyme substrate solution, I have bought TMB powder from Sigma but now i need the recipe and tips (i.e;TMB concentration, salt buffer composition, H202 concentration and other).

Thank in advance,

All the Best

We use a carbonate substrate buffer that we prepare in the lab for our pneumococcal polysaccharide and protein ELISAs. Not sure if it will be applicable to the kind of ELISA you're performing. The buffer comprises of two solutions, solution 1 contains 0.05M NaHCO3 (4.2005g in 1000ml ddH2O) and solution 2 contains 0.05M Na2CO3 (5.2995g in 1000ml ddH2O). Combine both solutions until pH is 9.8 then add 200mg of MgCl2.6H2O. In our enzyme substrate step, we add pNpp substrate tablets to the buffer (1 tablet/5ml buffer). Is yours an indirect ELISA? If so, the detection of Ab with conjugate should come after the test sample incubation step.

For coating: 0.1 mol/L carbonate buffer, pH 9.6

For washing: PBS, 0.3 mol/L NaCl + 0,1% v/v Tween 20 (= 0.01 mol/L phosphate buffer pH 7.2 + 0.3 mol/L NaCl, the doubled NaCl concentration helps to avoid unspecific reactions)

PBS: here Na/K: http://www.thelabrat.com/protocols/3.shtml

For incubation (sample and conjugate): washing buffer + 1 ... 3% on an inert protein like BSA, hydrolyzed gelatin, ....), add a pinch of phenol red (to see better where you are currently pipetting).

The substrate is available ready to use: http://www.seramun.com/products/substrates/horseradish-peroxidase.html

Stop: 0,1 mol/L H2SO4 (if you would like to measure much later you can add 0.01 mol/L sodium sulfide.

Product Description

3,3’,5,5’–Tetramethylbenzidine (TMB) is a chromogenic substrate suitable for use in ELISA procedures, which utilize horseradish peroxidase conjugates. This substrate produces a soluble end product that is blue in color and can be read spectrophotometrically at 370 or 655 nm. The reaction maybe stopped with 2 M H2S04, resulting in a yellow solution that is read at 450 nm.

Each tablet contains 1 mg of TMB substrate.

The product is available in packages of 50 or

100 tablets. Custom packaging and bulk purchase information available upon request.

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.

Preparation Instructions

0.05 M Phosphate-Citrate Buffer – Dissolve one Phosphate-Citrate Buffer Tablet (Product No. P 4809) in 100 ml of deionized water with stirring to yield a 0.05 M phosphate-citrate buffer, pH 5.0.

OR

Add 25.7 ml of 0.2 M dibasic sodium phosphate (Product No. S 0876), 24.3 ml of 0.1 M citric acid (Product No. C 7129), and 50 ml deionized water. Adjust pH to 5.0, if necessary.

TMB Substrate Solution – Dissolve one 3,3’,5,5’–tetra- methylbenzidine tablet in 1 ml of DMSO and add to 9 ml of 0.05 M Phosphate-Citrate Buffer, pH 5.0. Add 2 μl of fresh 30% hydrogen peroxide (Product No. H 1009) per 10 ml of substrate buffer solution, immediately prior to use.

OR

Dissolve one 3,3’,5,5’–tetramethylbenzidine tablet in 1 ml of DMSO and add to 9 ml of 0.05 M phosphate- citrate buffer, pH 5.0, containing 0.03% sodium perborate (capsules, Product No. P 4922).

Stop Solution - Reaction may be stopped by the addition of 50 μl of 2 M H2SO4 per 200 μl of reaction mixture.

Storage/Stability

Store the TMB tablets at 2–8 °C. Protect from heat, light, and moisture. Allow tablets to reach room temperature prior to use.

References

Bos, E., et al., J. Immunoassay, 2, 187 (1981).

PCS,MAM 01/05-1

The above is a PDF online from Sigma. Type in TMB solution recipe in Google.

I use 0,05M citrate buffer (0,05M citric acid adjusted to pH 3,8 with 6M NaOH - for prolonged storage I add 0,4% Kathon as preservative or freeze buffer or sterilize it in autoclave). To 15ml citrate buffer I add 150ul TMB solution (1mg/ml in mixture DMSO-ethanol 1:1) and 100ul 3% hydrogen peroxide (or 0,015% sodium perborate). With adjustment of buffer pH we will change sensivity of TMB substrate (at pH 3,8 sensivity are higher).  

Hi

best way

Bos, E., et al., J. Immunoassay, 2, 187 (1981).

PCS,MAM 01/05-1

Hello everyone,

I'm using ELISA for plant viruses first time and following DSMZ protocol. I need to prepare some buffers called

1. PBS-Tween:PBS+0.5 ml Tween 20/liter (this much only given in the protocol). I need to know how much PBS to be used in 1 L and what is that 1L is H2O or the PBS itself.

2. Sample extraction buffer: PBST+2% PVP

How much of PBST and 2% PVP in what quantity.

3.Conjugate buffer: PBST+2%PVP+0.2%egg albumin

how much of PBST, 2% PVP and 0.2% egg albumin in what quantity.

Can anyone help me out to prepare above buffers.

Thanks Chandrika

Hi, Emmanual, you can use the following protocol for development of ELISA:

Procedure for Use

1. Prior, to use, dissolve 5mg of OPD or one tablet in 9mL of water. Add 1mL of Stable Peroxide Buffer (Product No. 34062) to the dissolved OPD. Alternatively, dissolve OPD to 0.5-1.0mg/mL in a buffer containing 0.05M citric acid, 0.05M sodium phosphate; pH 5 and then add 1µL of 30% hydrogen peroxide per 1mL of substrate.

2. Add 100µL of the OPD solution to each microplate well and incubate for 10-30 minutes.

3. For non-stopped assays, measure the absorbance at 450nm.

4. To stop the reaction, add 50-100µL of 2.5M sulfuric acid to each well and measure the absorbance at 490nm.

Good luck...