* Alternatively, this assay can be run over two days, which requires 2 hours 30 mins (day 1) and 4 hours (day 2).
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
* Alternatively, this assay can be run over two days, which requires 2 hours 30 mins (day 1) and 4 hours (day 2).
PRODUCT SUMMARY
R&D Systems Cell-Based ELISAs simultaneously measure the levels of two proteins in the same microplate well in whole, fixed cells, eliminating the need for lysate preparation. These simple, sensitive assays can be used with both adherent and non-adherent cells to analyze protein phosphorylation or assess total protein levels.
Cell-Based ELISA Kits are available in two formats: phospho-protein kits contain antibodies to measure both the phosphorylated and the total protein, while total protein kits contain antibodies to the protein of interest and a second housekeeping protein. These two proteins are simultaneously detected in the same microplate well, allowing signals derived from the target protein to be normalized to that of the second protein. This permits corrections for well-to-well variation and allows target protein levels to be accurately assessed and compared across multiple samples. This Phospho- Cell-Based ELISA Kit employs fluorometric detection that is compatible with standard microplate fluorometers.
PRODUCT FEATURES
No lysate preparation required
Results with as few as 10,000 cells per well
Measure two proteins simultaneously in the same well
Ideal for measuring phosphorylation
Design allows for normalization of well-to-well variations
Complete kits require minimal optimization
Suitable for high-throughput analysis of non-adherent cells
Excellent alternative to Western blot and/or sandwich ELISA
KIT CONTENTS
96-well Microplate
Primary antibody for target protein
Primary antibody for normalization protein
HRP-conjugated secondary antibody
AP-conjugated secondary antibody
HRP Substrate F1
AP Substrate F2
Blocking Buffer
Wash Buffer
Plate Sealers
OTHER REAGENTS REQUIRED
37% formaldehyde (Molecular Biology Grade; Sigma, Catalog # F8775), refer to MSDS prior to use
30% H2O2 (Sigma, Catalog # H1009). Refer to MSDS prior to use
1X PBS (Irvine Scientific, Catalog # 9240)
Deionized or distilled water
Pipettes and pipette tips
Multi-channel pipette for washing
Cell culture incubator
Microfuge tubes
Orbital shaker
Fluorescence plate reader with two channels: excitation 540 nm/emission 600 nm and excitation 360 nm/emission 450 nm