Human Phospho-CBL (Y774) and Total CBL ELISA

Cell Lysates
Tissue Lysates

Colorimetric

96-well Microplate

Pre-
Coated 96-well Strip Microplate
Wash Buffer
Anti-
Phospho Antibody
H
R
P-
Conjugated Secondary Antibody
Assay Diluent
T
M
B One-
Step Substrate
Stop Solution
Lysis Buffer
Positive Control Sample

Prepare all reagents and samples as instructed in the manual.
Add 100 µl of sample or positive control to each well.
Incubate 2.5 h at R
T or O/
N at 4 °
C.
Add 100 µl of prepared primary antibody to each well.
Incubate 1 h at R
T.
Add 100 µl of prepared 1
X H
R
P-
Streptavidin to each well.
Incubate 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.

E3 ubiquitin-protein ligase CBL, Casitas B-lineage lymphoma proto-oncogene, Proto-oncogene c-Cbl, RING finger protein 55, RING-type E3 ubiquitin transferase CBL

RayBio® Phospho-CBL (Y774) and Total CBL ELISA kit is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in human cell lysates. By determining phosphorylated CBL protein in your experimental model system, you can verify pathway activation in your cell lysates. You can simultaneously measure numerous different cell lysates without spending excess time and effort in performing a Western Blotting analysis.This Sandwich ELISA kit is an in vitro enzyme-linked immunosorbent assay for the measurement of human phospho-CBL and total CBL. An anti-pan CBL antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and CBL present in a sample is bound to the wells by the immobilized antibody. The wells are washed and rabbit anti-CBL (Y774) antibody is used to detect phosphorylated CBL or rabbit anti-CBL antibody is used to detect pan CBL. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of CBL (Y774) or pan CBL bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

K562 cells were untreated or treated with PV. Cell lysates were analyzed using this phosphoELISA and Western Blot.