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Scientific Background
A reagent kit that allows our customers to develop their own cell-based ELISA. Determination of relative protein content and signal pathway (e.g. phosphorylation status) in whole cells. Evaluation of target modulation by drugs, activators, inhibitors, siRNA etc. All that is required is a primary mouse antibody (Ab1) for the specific target of interest. Key Features: New and improved. Total assay time reduced from the standard 21 hours to 6.5 hours (hands-on time 2.5 hrs). Simple and convenient. Cells are directly cultured in 96-well plates. No cell lysis necessary. Accurate and high-throughput.
Assay Principle
This Cell-Based ELISA Kit measures EnzyFluo™ Custom Cell-Based phosphorylation status directly in intact, fixed cells cultured in a 96-well plate — no cell lysis required. Cells are fixed, permeabilized, and incubated with a phospho-specific primary antibody followed by an HRP-conjugated secondary antibody. A fluorogenic substrate provides a signal proportional to phosphorylation level, normalized to total cellular protein measured in the same well to minimize well-to-well variation.
Key Features
- New and improved. Total assay time reduced from the standard 21 hours to 6.5 hours (hands-on time 2.5 hrs).
- Simple and convenient. Cells are directly cultured in 96-well plates. No cell lysis necessary.
- Accurate and high-throughput. Target protein is normalized to total cellular protein in the same well, greatly minimizing well-to-well variations. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.
Performance Specifications
| Total Assay Time | 6.5 hours (hands-on: 2.5 hours) |
|---|---|
| Compatible Sample Types | Cells (whole cells in 96-well plate) |
| Species Reactivity | Human, Mouse, Rat |
| Detection Method | Fluorometric/Colorimetric (FL530/585 nm, 360/450 nm) |
| Storage | -20 °C |
✓ Research-Grade Validation
Safety & Regulatory
Handle reagents in accordance with institutional biosafety guidelines. Refer to the Safety Data Sheet (SDS) for complete hazard and handling information. Contains components that may require special disposal procedures per local regulations.
This kit is validated for use with Cells (whole cells in 96-well plate). For unlisted matrices (e.g., tissue lysate, urine), perform a spike-and-recovery experiment to confirm assay performance before generating reportable data. Sample dilution in the kit's provided diluent is recommended to minimize matrix interference.
The minimum detectable concentration (sensitivity) of this kit is specified in the kit datasheet. Values below this threshold should be reported as below the limit of detection (<LOD) and should not be extrapolated from the standard curve.
The total assay time from sample addition to absorbance reading is approximately 6.5 hours (hands-on: 2.5 hours), including all incubation, wash, and substrate steps. Hands-on time is typically 1–2 hours; most steps involve passive plate incubation. Plan the assay as a single uninterrupted session for best results.
Standard components of this Cell-Based ELISA Kit typically include: pre-coated microplate (96-well strip format), lyophilized or liquid recombinant EnzyFluo™ Custom Cell-Based standard, detection antibody, streptavidin-HRP conjugate, TMB substrate, stop solution, wash buffer concentrate, and sample/standard diluent. Refer to the kit insert or datasheet for the exact component list and storage requirements.
This kit uses colorimetric (TMB/HRP) detection and requires a standard microplate absorbance reader capable of measuring at 450 nm. A reference wavelength of 570 nm or 630 nm is recommended to reduce background. No specialized fluorescence or luminescence reader is needed. Ensure the instrument is calibrated and the plate is clean and free of condensation before reading.
Can’t Find What You’re Looking For? We can help you source the best match or customize an ELISA solution for your study. Options may include alternative target synonyms, different species reactivity, sample type/matrix compatibility (serum/plasma/lysate/supernatant), assay format (sandwich/competitive), sensitivity/range, detection chemistry (colorimetric/fluorescent/chemiluminescent), plate format (pre-coated/uncoated, strips vs full plate), and bulk or custom packaging. Click Talk to a Scientist to submit a request form, email us at support@biohippo.com, or explore our Research Services for additional support. Our team will be in contact with you shortly.
