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Tmb substrate solution

Manufactured by R&D Systems

TMB substrate solution is a laboratory reagent used in various immunoassay techniques, such as enzyme-linked immunosorbent assays (ELISAs). It serves as a chromogenic substrate for the detection and quantification of enzyme-labeled target molecules. The solution contains 3,3',5,5'-tetramethylbenzidine (TMB) and other proprietary components.

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2 protocols using tmb substrate solution

1

Quantification of Allergy Biomarkers in BAL Fluid

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The levels of IL-5 and IL-13 in BAL fluid were measured by Quantikine and Duoset ELISA kits (R&D Systems) according to the manufacturer’s instructions. Total IgE was determined by the Clonotyping ELISA kits (Southern Biotech). Ovalbumin-specific IgE in mouse sera was quantified as following. Immulon 2HB plates were coated with 2 μg ovalbumin in bicarbonate buffer (pH 9.6) per well overnight at 4 °C and blocked with 1% BSA. Serum samples were diluted in 0.1% Tween 20-PBS and incubated for 2 hours at room temperature. OVA-specific-IgE was detected by using goat-anti-mouse IgE conjugated with HRP (Southern Biotech). Plates were developed with TMB substrate solution (R&D Systems) and reactions were stopped with 1N HCl. Absorbance values at OD450 were measured. Concentrations of ovalbumin-specific IgE were determined using a standard curve made with serum from hyper-immunized mice.
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2

Plasma Biomarker Detection by ELISA

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Three mL of plasma was isolated by centrifuging the tubes at 250 g for 10 minutes. The wells of a 96-well microplate were coated with capture antibody prolactin (PRL) (R&D, America), placenta growth factor (PIGF) (R&D, America) or nerve growth factor receptor (NGFR) (R&D, America) with 30 µL of coating buffer (0.1 M sodium carbonate-sodium bicarbonate, PH 9.5; 1.59 g Na2CO3 and 7.13 g NaHCO3 in 1 L dH2O) overnight at 4 °C, and were removed the coating buffer for adding 50 µL blocking buffer (1 mL PBS) at RT BSA%. Protein samples (30 µL) were pre-mixed with dilution buffer (PBST, 30 µL PBS, PH 7.4, containing 0.05% Tween 20, 1% BSA and 5 µg protein) for 1 h. The mixture was loaded and incubated for 1 h at RT, and the plate was washed three times with PBST. Next, 30 µL of the detection antibody (R&D, America) in the dilution buffer was separately added. The plates were incubated for 1 h at RT and washed three times with PBST. The reaction was observed by adding 30 µL/well of TMB substrate solution (R&D, America) at RT for 30 minutes. The reaction was stopped with 30 µL/well of stop solution (NaH2SO4). ELISA plates were detected at 450 nm using an Epoch microplate spectrophotometer (Bio-Tek).
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