R&D Systems Cell-Based ELISAs are the first two-color immunoenzymatic
assays to permit the simultaneous measurement of both phosphorylated and total
proteins in the same microplate well without the lysis of cells. Normalizing
the fluorescence signal derived from the phospho-protein to that of the total
protein in the same well allows for the accurate correction of well-to-well
variabilities such as differences in cell number.
Advantages
- No lysate preparation
- Results with as little as 10,000 cells/well
- Measures total and phospho-proteins simultaneously in the same well
- Amenable to high-throughput screening of kinase inhibitors
- Correlates with Western blot
Current Cell-Based ELISA Kits Available
View Larger Image
|
Figure 1. Cells are fixed and first incubated with anti-phospho and anti-total
antibodies derived from different species. Alkaline phosphatase (AP)- and
horseradish peroxidase (HRP)-conjugated secondary antibodies recognizing
the different primary antibodies are then added, followed by incubation
with two spectrally-distinct fluorogenic substrates that emit signals corresponding
to the levels of phosphorylated and total proteins. |
View Larger Image
|
Figure 2. Phosphorylation of
EGF R was determined in A431 epidermoid carcinoma cells using R&D
Systems human Phospho-EGF R (Y1068) Cell-Based ELISA Kit (Catalog # KCB1095).
Cells were cultured in 96-well plates and treated with increasing concentrations
of R&D Systems human EGF (Catalog # 236-EG) for 5 minutes. After
cell fixation, EGF R phosphorylation was determined and normalized to
total EGF R in the same well. Values represent the mean ± the
range of duplicate determinations. A comparison Western blot is also
shown (inset). |
View Larger Image
|
Figure 3. Effects of the EGF R tyrosine kinase
inhibitor PD168393 on EGF-induced EGF R phosphorylation were measured using
the human Phospho-EGF R (Y1068) Cell-Based ELISA Kit (Catalog # KCB1095).
A431 epidermoid carcinoma cells were pretreated for 30 minutes with the
indicated concentrations of PD168393. Cells were then incubated with no
additions or with 50 ng/mL (5 min) EGF (Catalog # 236-EG) and fixed. Phosphorylation
of EFG R was determined and normalized to total EGF R in the same well.
Values represent the mean ± the range of duplicate determinations. |