Human/Mouse/Rat Phospho-Tyrosine Cell-Based ELISA

Human

Cell-based

Semi-Quantitative

Cell Culture Microplate
Wash Buffer A
Wash Buffer B
Fixing Solution
H
R
P-
Conjugated Secondary Antibody
Blocking Buffer
T
M
B One-
Step Substrate
Stop Solution
Quenching Buffer

Seed 10, 000-30, 000 cells into each well and incubate overnight.
Apply various treatment, inhibitors or activators according to manufacture's instructions.
Add 100 µl of Fixing Solution into each well and incubate for 20 min at R
T with shaking.
Add 200 µl of prepared 1
X Quenching Buffer and incubate 20 min at R
T.
Add 200 µl of Blocking Solution and incubate for 1 h at 37 °
C.
Add 50 µl of 1
X anti-phospho-protein specific antibody or anti-pan-protein specific antibody to each well and incubate for 2 h at R
T.
Add 50 µl of prepared 1
X H
R
P-
Anti-
Rabbit or Mouse Ig
G and incubate for 1 h at R
T.
Add 100 µl of T
M
B One-
Step Substrate Reagent to each well.
Incubate 30 min at R
T.
Add 50 µl of Stop Solution to each well.
Read at 450 nm immediately.