Accurate Quantification
Wide linear dynamic range
Multiplex Detection
Normalization increas quantification accuracy
High Sensitivity
Equal to or better than chemiluminescence
Direct Detection
Analyzes multiple targets in 96- or 384-well plates.
Immunofluorescent-Bad Assay
Fast, microplate-bad assay. Lysate preparation, gel loading,
electrophoresis, and membrane transfer are eliminated.
Multiple Targets
Ideal for screening cell treatments or drug candidates
for effects on target proteins.
In-Cell Western
™
QuantItatIve Cell-Bad assays
In-Cell Western Assays offer broad application to the analy-sis of protein signaling pathways, reliable
protein quantifica-tion, and cell-bad determinations of IC 50 concentrations for lead optimization. In-Cell Western Assays are also a powerful tool in the study of the effects of drug components on multiple points within one or more signaling pathways.
Other traditional protein assay methods such as Western blotting are cumbersome and labor intensive. High content screening methods employ very expensive complex instrumentation and consumable costs. In-Cell Western Assays offer a practical alternative for medium to high throughput analysis.
In-Cell Western Assays simultaneously detect two targets at 700 and 800 nm using two spectrally distinct dyes. Separate lars and fluorescence detectors are ud for each dye and offer a wide linear detection range.
With two detection channels you can probe two parate targets or increa quantification accuracy by using the cond channel for normalization. Quantification accuracy is maximized by normalization becau adjustments can be made for differences in cell number from well to well. Two-color normalization also helps prevent fal negatives and provides more accurate evaluation of cell treatment or drug candidate effects.
800 nm (Phospho-ERK)
700 nm (Total ERK)
Composite Image Overlay
U0126
eGFR signaling
Signaling by the Ras/Raf/MEK/ERK pathway was activated in A431 cells by stimulation of the epidermal growth factor receptor (EGFR)
with EGF . Cells were also treated with drug
compounds (blue arrows) that inhibit the signaling cascade at two different points. PD168393 is a known EGFR inhibitor and should affect not only EGFR auto-phosphor-ylation but also downstream targets such as STAT3 and ERK1/2. In contrast, U0126 is an inhibitor of MEK1/2. This drug should inhibit signaling by MEK1/2 and cau a decrea in ERK1/2 phosphorylation, without affecting phosphorylation of EGFR or STAT3.
1401201008060402000
Concentration PD168393 (nM)
% P h o s p h o r y l a t i o n
A
B
Concentration U0126 (µM)
% P h o s p h o r y l a t i o n
pEGFR pERK pStat3
pEGFR pERK pStat3
