Overview and Menu of In-Cell ELISA Kits

Monitor relative protein levels and post-translational modification by ELISA using whole cells.

The target-specific Thermo Scientific In-Cell ELISA Kits are optimized systems for accurate quantitation of relative protein expression levels using either colorimetric or fluorescent near infrared detection methods.

The target-specific In-cell ELISA Kits are complete and highly effective cell-based assays for characterizing specific proteins in whole cells. Each kit includes the detection reagents, specific antibodies, buffers and optimized procedure to measure two realative protein amounts such as p53 and Tubulin or STAT6 and phosphorylated STAT6. The assays are performed in 96- or 384-well microplates, easily scalable and conserve cell culture and treatment reagents. Furthermore, the assays are amenable to automation and statistical analysis, which is ideal for screening drugs.

Page Contents:

• In-Cell ELISA Kit Detection Methods
• Overview of In-Cell ELISA Kit Protocol
• In-cell ELISA vs Western blotting
• Multiplex Assays with In-Cell ELISA Kit
• Menu of In-Cell ELISA Kits

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Overview of ELISA

In-Cell ELISA Kit Detection Methods

The Thermo Scientific In-Cell ELISA Kits were developed to permit accurate quantitation of relative protein expression levels. These kits are available in two different formats, one for use with colorimetric plate readers and one format for use with near infrared imaging systems. Target-specific In-Cell ELISA Kits can be purchased in either format and the key features of each are described below.

• Colorimetric Detection Method – The Colorimetric In-cell ELISA Kits enable multiple target protein levels to be compared from different wells using target-specific antibodies, HRP-conjugated detection reagents and TMB substrate. The results across each well are then normalized to cell number based on whole-cell staining with Janus Green.
• Near IR Detection Method – The Near Infrared In-Cell ELISA Kits enable the simultaneous detection of two different target proteins within the same well, eliminating the variability caused by differences in cell plating. With the near IR system, protein levels are determined by using target-specific primary antibodies and DyLight Fluor-Conjugated Secondary Antibodies. The near IR detection method eliminates the issues with background fluorescence, providing a high signal-to-noise ratio and making these fluorescent multiplex experiments both sensitive and robust.

Overview of the Thermo Scientific In-Cell ELISA Kit Protocol

The colorimetric and near infrared detection methods allow multiple targets proteins to be analyzed in a single experiment. The use of fluorescently-tagged antibodies allows two targets to be compared within the same microplate well with the Near Infrared Detection Method. When using the Colorimetric Detection Method, different proteins are measured in separate wells and then the data is normalized by the cell number in each well using the whole-cell stain Janus Green.

In-cell ELISA vs. Western blotting

Cell signaling pathways involve changes in the post-translational modification and expression levels of proteins. These changes are typically measured by relative comparison to the levels of house keeping proteins such as GAPDH, Actin, etc. by Western blotting. While a powerful tool for analyzing protein expression, Western blotting is only semi-quantitative, has low throughput and does not easily permit statistical measurements.

Although the same results can be obtained from a Western blotting experiment and In-cell ELISA assays, the In-cell ELISA has several key advantages over blotting experiments. Most notably, it is much more convenient to analyze replicates for statistical analysis using the plate-based ELISA method. For large experiments, up to 96 or 384 sample can be read quite easily from a single microplate. Even a much smaller experiment analyzed by Western blotting would require a different gel for every 15-20 samples. Once developed, each lane in a Western blot must be analyzed by densitometry requiring significantly more time to process than and ELISA microplate.

In-cell ELISA provides advantages over Western blotting. Dose-dependent activation of STAT3 and EGFR by EGF was evaluated in A431. Panels A and B: Phospho-STAT3 and phospho-EGFR increased with increasing doses of EGF as determined by the Pierce In-Cell ELISA Kit (A) and Western blot (B).

Multiplex Assays with In-cell ELISA Kits

Multiplex analysis is possible with both the colorimetric and near infrared In-Cell ELISA Kits. With the colorimetric detection method, differences in protein levels are compared in different wells and then normalized by cell number. With the near IR detection method, two targets can be measured in the same well using the two respective fluorescent channels. Regardless of the method used, both types of In-Cell ELISA Kits provide the same results.

The Colorimetric and Near Infrared In-Cell ELISA Kits are fast and reliable formats for determination of protein activation and inhibition. Evaluation of dose-dependent activation and inhibition of EGFR in A431 cells with EGF and PD169393. Panels A and B show an increase in phospho-EGFR with increasing doses of EGF. The EGF-induced phosphorylation of EGFR is inhibited with increasing concentrations of PD169393 (panel C, rows C and D), whereas total EGFR does not significantly change with either treatment. No significant staining is seen in the absence of primary antibodies (panel A, column 1 and panel C, column 12). Panels B and D show that the fluorescence (RIU, left Y-axis) values obtained with the near infrared detection method can be overlaid on top of the data obtained with the colorimetric detection method (right Y-axis).

Target-Specific In-Cell ELISA Kits

Related Products:

• In-Cell ELISA Detection Reagent Kits – basic reagent kits for use with any primary antibody
• Target-specific In-Cell ELISA Kits – browse kits for specific targets