In-Cell ELISA Kits

A quantitative alternative to western blot

In-cell ELISA is an accurate method to determine the relative protein levels and degree of post-translational modification (PTM) in whole cells of various types. By contrast, traditional western blot analysis is time-consuming and only semi-quantitative.

Thermo Scientific™ In-Cell ELISA Colorimetric and Near Infrared Detection Kits enable easy quantitation of intracellular protein levels in whole cells. The kits enable the in-cell ELISA method to be performed in 96- or 384-well microplates, are more easily scalable than western blotting, and conserve cell culture and treatment reagents. Quantitative results are easily analyzed because they are recorded using a standard ELISA plate reader or infrared imaging system. Also, the plate-based procedure is amenable to automation.

Applications

  • High throughput determination of relative protein expression levels
  • Monitor dose-dependent post-translational protein modification
  • Multiplex analysis of targets in multiple cell lines

Features of In-cell ELISA

  • Simple, fast assay format
  • Ability to perform multiplex experiments
  • No special sample preparation required
  • Higher throughput compared with western blotting
  • Two detection formats available
    • Near Infrared–compatible with the LI-COR™ Odyssey Imaging System
    • Colorimetric–compatible with standard ELISA plate readers

Quantitative advantages over western blotting

Comparison of the In-Cell ELISA method with traditional western blotting.

  In-cell ELISA Traditional western blot
Quantitative Yes No
Relative throughput High (96- or 384-well plates) Low (limited by gel lanes)
Assay time 4 hr 8-24 hr
Strip and reprobe Yes Yes
In-cell ELISA provides advantages over western blotting.
In-cell ELISA provides advantages over western blotting. Dose-dependent activation of STAT3 and EGFR by EGF was evaluated in A431 cells. Phospho-STAT3 and phospho-EGFR increased with increasing doses of EGF as determined by (A) Thermo Scientific™ In-Cell ELISA Kits and (B) western blot analysis.

Two protocol and kit options for In-cell ELISA detection

Near Infrared In-cell ELISA—These 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™ fluorophore-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.

Colorimetric In-cell ELISA—These 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 and colorimetric detection methods of the Thermo Scientific In-Cell ELISA Kits

Schematic overview comparing the near IR and colorimetric detection methods of the Thermo Scientific In-Cell ELISA kits. The In-Cell ELISA Near Infrared Detection Kit allows two targets to be analyzed in the same well using two primary antibodies and detection with DyLight 680 and DyLight 800 conjugated secondary antibodies. The In-Cell ELISA Colorimetric Detection Kit allows multiple targets to be analyzed using primary antibodies in different wells detected with a horseradish peroxidase conjugate normalized to cell number with the whole-cell stain, Janus Green.


Near IR and colorimetric In-Cell ELISA kits produce similar results

 

Near IR and Colorimetric In-Cell ELISA Kits produce similar results. The Thermo Scientific In-Cell ELISA Near Infrared Detection Kit and the Thermo Scientific In-Cell ELISA Colorimetric Detection Kit were compared using EGFR activation in A431 cells as the model system. Similar fold-induction (see graph) and Z-factor (0.82 and 0.80, respectively) indicate comparable and robust performance of both kits.


EGF near IR in-cell ELISA results
EGF near IR and colorimetric in-cell ELISA results
PD168393 near IR in-cell ELISA results
PD168393 near IR and colorimetric in-cell ELISA 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).


Multiplex in-cell ELISA results for STAT3, STAT6, EGFR
Multiplex in-cell ELISA results for STAT3, STAT6, EGFR

Multiplex analysis of multiple targets using the In-Cell ELISA Near Infrared Detection Kit. Levels of total and phosphorlylated STAT3, STAT6 and EGFR were measured in untreated A431 cells and compared to cells treated with 100 ng/mL EGF or both 100 ng/mL EGF and 25 nM PD168393 (panel A). Graphing the data shows inhibition but not complete blocking of phosphorylation by 25 nM PD168393 (panel B).


Generic and target-specific kits available

Choose from one of the basic In-Cell ELISA Kits or from the target-specific In-Cell ELISA Kits for characterizing specific proteins in whole cells. The target-specific In-Cell ELISA Kits are complete kits that include the detection reagents, specific antibodies, buffers and optimized procedure to measure two relative protein amounts such as p53 and tubulin or STAT6 and phosphorylated STAT6.

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