Kpl tmb microwell peroxidase substrate system

We established a Sandwich ELISA system to identify NUDT15 protein with
high specificity. In brief, 50 μl of the capture Ab (clone #1, 7, 10
μg/ml) in ELISA Coating Buffer (Abcam) was added to each well of a
96-well ELISA plate (Abcam) and the plate was allowed to coat for 1 h at RT. The
plate was washed three times with washing buffer (WB; 1 × PBS containing
0.05% Tween-20) and then blocked with 300 μl of 1 × Blocking
Buffer (Abcam) for 1 h at RT. Then, plates were washed three times with WB and
50 μl samples or standard NUDT15 protein was purified as previously
described⁸. Then, they were added and incubated for 1 h at RT.
Next, plates were washed three times with WB and 50 μl of biotinylated
detection Ab solution (clone #4, 10, 1 μg/ml in 1 × Blocking
Buffer, biotinylated using EZ-Link™ Sulfo-NHS-LC-Biotinylation) was added
and plates were incubated at RT for 15 min. Then plates were washed three times
with WB, and 50 μl of Streptavidin-Horseradish Peroxidase (HRP) Conjugate
(Thermo Fisher Scientific) diluted 1 : 8000 in blocking buffer was added and
incubated at RT for 15 min. The plates were washed three times with WB, and 100
μl of a two-component TMB substrate system (KPL TMB Microwell Peroxidase
Substrate System, SeraCare) was added to each well. After 10 min of incubation
at RT, 100 μl of Stop Solution (Abcam) was added and absorbance was
recorded at 450 nm.