TMB Solution

Home-Made TMB Solution

Working Protocol 

Solution A:

Dissolve

Citric acid monohydrate: 3.60 g

trisodium citrate dihydrate: 1.838 g

in 950 ml of double-distilled water.

Add 0.564 g Urea hydrogen peroxide while continuous stirring.

Solution B

Add 100 mg TMB to 15 ml  N,N-Dimethylacetamide. Store in a dark bottle at -20 oC.

Working Solution

Just before use, add 50 ul solution B to 5 ml Solution A, then add 1 drop of 30% H2O2.

21:50, 5 April 2018 (EST)~

Phosphate-Citrate Buffer 0.1 M at pH 4.6 ± 0.2 plus perborate

0.2 M dibasic sodium phosphate

Dissolve 1.78 g Na2HPO4-2H2O  or 3.58 g of Na2HPO4•12H2O in dH2O

Add  26 ml 0.1 M citric acid (2.10 g citric acid monohydrate in dH2O and make to 100 ml with dH2O)

Add H2O to 50 ml.

Check pH

Dissolve 0.02 g sodium perborate.

Store in polyethylene bottle at 4 °C stable 1 year.

TMB substrate 100 mM

Dissolve 240 mg 3,3’,5,5’–tetramethylbenzidine (m.w. 240) in 10 ml of DMSO. Store in opaque polyethylene bottle at 4 °C stable 1 year.

Working TMB chromogen Mix 25 µl of TMB substrate to 1.0 ml of phosphate-citrate buffer plus perborate, stable for several hours at room temperature or for few days at 4 °C.

Patent

https://patents.google.com/patent/US5206150

Dissolve

Citric acid monohydrate: 3.60 g

trisodium citrate dihydrate: 1.838 g

in 950 ml of double-distilled water.

Add 1.422 g Bacitracin with stirring until it dissolved.

Add 0.564 g Urea hydrogen peroxide while continuous stirring.

Add 300 mg 3,3',5,5'-Tetramethylbenzidine (TMB) to 50 ml of dimethyl sulfoxide

Add TMB solution to aqueous solution.

Mixing in an amber bottle

Store at 4° C.

The final concentrations of each of the above components is: bacitracin, 1.0 mM; urea hydrogen peroxide, 6 mM; TMB, 1.25 mM; DMSO, 5%; citrate, 25 mM and the final pH is 3.4.

Protocol from  Journal of Immunological Methods 233 2000. 47–56

Buffer A

205 mM potassium citrate, pH 4.0, 3.075 mM H2O2 . was freshly prepared before each experiment from sterile-filtered pore size 0.22 mm.
205 mM potassium citrate, pH 4.0, and 30% H2O2, both stored in polypropylene or polyethylene containers. For the analysis of long-term stability, Buffer A was kept in polypropylene containers at 4oC.

TMB/DMA (41 mM TMB in DMA. absolute, H2O-content : 0.01%), N,N-Dimethylacetamide

Working Solution

Prepare 10 to 20 min before use by adding 200 ml Solution B or TMB/DMA or TMB/DMSO. to 8 ml Buffer A. 

Add H2O2 to a final concentration of 1.3 mM immediately before use