A 96 well-plate was incubated with rhFGFR1α(IIIc) (100 μL per well at a concentration of 0.5 μg/mL in D-PBS) for 12 hours at 23 °C. After 16 hours the wells were washed and then blocked with 400 μL of 1% bovine serum albumin (BSA) in D-PBS (2 hours). Next, sample solutions were prepared at 1 ng/mL and aliquoted into Eppendorf tubes. After washing the wells, 100 μL of the sample solutions were added to each well. For protein solutions, heparin or polymer was added so that the final concentration of heparin or polymer was 1 μg/mL. After 2 hours the wells were washed and 100 μL of FGF2 antibody-biotin conjugate was added and incubated for an additional 2 hours before washing and incubating with streptavidin-horseradish peroxidase solution for 20 minutes. The plate was developed by incubating with 100 μL of 1-Step Ultra 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Pierce Biotechnology, Rockford) for 8 minutes. The assay was terminated by the addition of 50 μL of 1 M H2SO4. Absorbance was read at λ = 450 nm. Each sample was plated in triplicate.
1 step ultra 3 3 5 5 tetramethylbenzidine tmb solution
Manufactured by Thermo Fisher Scientific
The 1-Step Ultra 3,3′,5,5′-tetramethylbenzidine (TMB) solution is a ready-to-use substrate solution for the detection of horseradish peroxidase (HRP) in enzyme-linked immunosorbent assays (ELISAs) and other HRP-based colorimetric applications. The solution contains TMB, a chromogenic substrate that produces a blue color upon oxidation by HRP.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using 1 step ultra 3 3 5 5 tetramethylbenzidine tmb solution
1
FGF2 Receptor Binding Assay
2
FGFR1 Binding Assay Protocol
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A
96-well plate was
incubated with rhFGFR1α(IIIc) (100 uL per well at a concentration
of 0.5 ug/mL in D-PBS) for 16 h at 23 °C. After 16 h, the wells
were blocked with 1% bovine serum albumin (BSA) in D-PBS (2 h). Next,
solutions of protein or conjugate were prepared at a concentration
of 1 ng/mL, and 100 uL was plated in the wells and then incubated
for 2 h. After 2 h, 100 uL of FGF2 antibody–biotin conjugate
was added and incubated for an additional 2 h before streptavidin–horseradish
peroxidase solution was incubated for 20 min. The plate was developed
by incubating with 100 uL of 1-Step Ultra 3,3',5,5'-tetramethylbenzidine
(TMB) solution (Pierce Biotechnology, Rockford) for 8 min. The assay
was terminated by the addition of 50 uL of 1 M H2SO4. Absorbance was read at λ = 450 nm. Each sample was
plated in triplicate.
96-well plate was
incubated with rhFGFR1α(IIIc) (100 uL per well at a concentration
of 0.5 ug/mL in D-PBS) for 16 h at 23 °C. After 16 h, the wells
were blocked with 1% bovine serum albumin (BSA) in D-PBS (2 h). Next,
solutions of protein or conjugate were prepared at a concentration
of 1 ng/mL, and 100 uL was plated in the wells and then incubated
for 2 h. After 2 h, 100 uL of FGF2 antibody–biotin conjugate
was added and incubated for an additional 2 h before streptavidin–horseradish
peroxidase solution was incubated for 20 min. The plate was developed
by incubating with 100 uL of 1-Step Ultra 3,3',5,5'-tetramethylbenzidine
(TMB) solution (Pierce Biotechnology, Rockford) for 8 min. The assay
was terminated by the addition of 50 uL of 1 M H2SO4. Absorbance was read at λ = 450 nm. Each sample was
plated in triplicate.
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