Cell-Based ELISA Base Kit 1

* 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 ELISA Base Kits are designed to facilitate the detection of two proteins in the same microplate well in whole, fixed cells, eliminating the need for lysate preparation. Base kits include microplates, buffers, diluents and two enzyme-conjugated secondary antibodies that cleave two spectrally distinct fluorogenic substrates. The user simply adds the primary antibodies of choice.

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 Base Kits employ fluorometric detection that is compatible with standard microplate fluorometers. The 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.

PRODUCT FEATURES

• Add your own primary antibodies
• 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
• HRP-conjugated secondary antibody
• AP-conjugated secondary antibody
• HRP Substrate F1
• AP Substrate F2
• Blocking Buffer
• Wash Buffer
• Plate Sealers

OTHER REAGENTS REQUIRED

• Primary antibody for target protein
• Primary antibody for normalization protein
• 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