NEW TOOLS: CELL-BASED ELISAS

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 Human Phospho-EGF Receptor (Y1068) (Catalog # KCB1095) Human Phospho-ERK1/ERK2 (T202/Y204) (Catalog # KCB1018) Human Phospho-PDGF Rb (Y1021) (Catalog # KCB2316) Human Phospho-PDGF Rb (Y751) (Catalog # KCB1767)
	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.
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).	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.