Hi Jhoti

I have used TMB from commercial kits for several times and I have had not problems. Keep the TMB at 4°C. Do not frozen. Actually, I think your TMB is contaminated.

You should use the substrate directly within 10 min.

within a couple of minutes of preparing the TMB substrate, it started turning pale blue! 

The TMB solution needs to be kept cold and I would not pipette from the stock bottle. I would pour aliquots out into a disposable clean container and the use right away. If you dissolve a pellet in a solution to prepare your TMB, it should not have any peroxide in it.  Then store it in the cold. You probably have peroxide contamination somewhere, or something that will react with the TMB. I would buy the solutions already made, and keep that safe From contamination. If your stock solution is already made, and you dilute it into something and it turns blue right away, I would buy new material and/ or make sure you are working with clean surfaces and solutions.  When the TMB solution gets old, sometimes it will turn blue right away.

Hi,Somanah

if your TMB is blue, it exposur to light, you must keep TMB in dark bottle at 4C

Can TMB be frozen once made? Im using TMB from an ELISA kit (Biolegend Inc.)

Hi Jhoti

I have used TMB from commercial kits for several times and I have had not problems. Keep the TMB at 4°C. Do not frozen. Actually, I think your TMB is contaminated.

If you use the same vessel to dispense the TMB as the one you used to dispense the HRP, then it will turn blue quite fast. :)

Dear Jhoti,

This is a common problem with lab med prepared TMB solution.

As you know TMB can be oxidized by oxidizing agent and convert to blue product. Commercial TMB has stabilizer and prevent to change in blue color before its reaction with e.g HRP. but there is a simple and grantee secret solution. After preparing TMB in citrate buffer, add 0.1ml sodium thiosulfite 0.05% to 10ml TMB solution. It will prevent spontaneously blue color formation.

It is interesting if your TMB is blue now, you can add the magic solution, convert it to colorless solution and you can use it again.

I hope this simple secret help you.

best

mehdi 

This is most likely due to the peroxide aspect of the substrate.  If possible I would suggest going to a one component TMB substrate as this tends to be a bit more light stable.

when I did use two compoennt TMB I would pull an aliquot out of each bottle, wrap in foil and put in a drawer to allow it to come to room temperature.  Just before adding to the plate (usually while washing the plate) I would combine the reagents, mix by inversion and keep in foil wrapped tube.  once I had the plate back at my bench I would then pour it in a reservior and add to plate.

I also suggest incubating the plate in the dark (again, I usually put in a drawer) to keep the light blue color from appearing.

Great tip Mehdi.

THANKS for the tip Mehdi.  I have had some trouble with TMB on tissue sections (brain) with the deposits looking "clumpy" under high power.  This might help if I needed to start over.

You're welcome dear Rose.

Thanks for the advice guys. I should take extra care by using clean tips and plastic containers for mixing. 

You're welcome, yes it is important new or clean tips and other things during using TMB, but you can check the magic formula, it reduce the sensitivity about the vessels, tips,.. and will offer you a clean TMB substrate without blue background.

best

Hi, it sounds like either your TMB stock is old, or contaminated. If it is old you may see some precipitant too. There are different types, either made up or combining 2 solutions. Always mix it fresh before use.

Always store your TMB at 4C in a dark container. As other people mentioned always aliquot some or your stock in a new container to avoid contaminating your stock. Always do your incubations in the dark.

and one extra thing as an advice,

Most protocols stop the reaction using 2NH2SO4 or 1M H3PO4 which turns the reaction yellow, after a specific incubation time and read their plate (endpoint assays)

I don't tend to stop my reaction but I tend to take successive readings of the plate as sometimes the reaction may get saturated quite early.

hope that helps

As other researches have pointed out, in case of commercially available prepared - ready to use TMB ( Usually provided in  brown  bottles), always make sure the tips used  new. In case of Seperate Concentrate and buffers- make sure the containers used  are washed well and always seperate tips are used. and the solution should be freshly prepared just before use and not exposed to light. 

All my dear

yes, we know TMB cab be oxidize by oxidizing agent such as H2O2, persulfates,....

Enzymes such as HRP can be catalyze the reaction. Its product is oxidized TMB, with blue color which can polymerize to yellow to brown color in acidic media.

This is wanted reaction.

But unwanted reaction can be converted TMB to its blue product.

oxygen, light,...can speed unwanted reaction.

We know one step reagent (TMB+Oxidizing agent such as H2O2) is more unstable than two steps solution contains TMB and H2O2 separate.

So:

Use Two steps solutions instead of one step type.

Keep away from light in brown or foiled bottle.

keep in cold temperature

Keep from any oxidizing contamination.

Keep from any catalyzing contamination(Hb, HRP,...).

keep solution in suitable bottle with minimum empty space.

Use new tip for bypass contamination.

....

all items can help us, but do not forget:

With considering the all above items now it is possible unwanted reaction and appearing blue color.

So with help f smooth antioxidant or reducing agent we can prevent or reverse the oxidizing TMB unwanted reaction.

Finally use of the magic solution (thiosulfate solution) is a very good preventing and reversing agent for this problem.

best

mehdi

YOu have a contamination, either by the HRP itself or by socalled pseudoperoxidases. All proteins containg an Hem-ring with Fe++ or even Fe++ (rust) will catalyze the reaction. 

So make sure that you are using reaction bowls which have never seen anything from above. 

Ready to use substrates can be helpful here: 

http://www.seramun.com/produkte/substrate/elisa-substrate-fuer-meerrettich-peroxidase-pod.html

All my dear

The TMB Substrate Set (TMB Substrate A and TMB Substrate B) should be stored between 2°C and 8°C. Avoid prolonged exposure to light, contact with metal, air, or extreme temperatures.

Hi there, I just had this exact problem.  I went systematically through every step and reagent of an ELISA that I have performed for almost 10 years without a single problem, and was unable to pinpoint the culprit for quite a while.  I work in a compliance-regulated lab; we maintain our reagents to strict standards.  (2-part TMB stored cold, in amber bottles, prepared only at the time of use, and always protected from light.)

Despite this, I found, across multiple brand new bottles from the same lot and two new lots, several hot wells and premature color change of the substrate in brand new, individually wrapped reagent reservoirs.  There was no way these bottles were contaminated, and yet, the problem persisted.

Here's the thing that I didn't know until I called the TMB manufacturer's technical services: TMB can exhibit premature color change in the presence of metallic ions.

Our cafeteria, located immediately beneath my lab, was being renovated.  Lots of dust, metal studs being cut.  The problems I had with my TMB started immediately after construction began, and, now five months later, construction has finished and I can't duplicate the problem again even when I go back to stock bottles that repeatedly demonstrated this issue in the past.

It wasn't the substrate bottles, and it wasn't me.  Everything points to airborne contaminants.

If it helps, this is mentioned in passing in this troubleshooting guide (page 9): https://resources.rndsystems.com/pdfs/datasheets/edbapril02.pdf

HI everyone, I have a question related somewhat to this discussion. I am preparing a std curve of Cytochrome C. While dispensing the std solution to TMBZ, it turns dark brown and then i hv to add PBs followed by Hydrogen peroxide. but instead of blue the color of reaction mixture turns to light brown after 30 mins of incubation at room temperature. TMBZ solution was made by dissolving 0.01 g of TMBZ in 5 ml of

methanol and 15 ml of 0.25 M sodium acetate buffer (pH 5.0). Please suggest me the probable cause behind this. I am stuck in the expt.

Perhaps due to enhanced oxidation

Sir, is it because TMBZ is not working properly, or reaction between Cytochrome C and TMBZ is not taking place normally..

I am not sure your experiment requires reduced or oxidized form of cytochrome c. Preparation of oxidized cytochrome c may not be tricky, but reduced form of cytochrome c  can be prepared utilizing either sodium dithionite or sodium ascorbate, followed by gel filtration.

1. Nature, 213(74), 399-400 (1967).

2. Methods in Enzymology, 53D, 40-47 (1978).

 To Jhoti:

Your TMB substrate should be clear before adding to wells. You are correct, light exposure may have oxidized TMB.

Sounds more like you have some contamination