In-cell ELISAs for measuring relative protein expression and modification levels

The Thermo Scientific In-Cell ELISA Colorimetric and Near Infrared Detection Kits are simple and convenient methods for quantification of intracellular protein levels in whole cells. While traditional Western blot analysis is time consuming and only semi-quantitative, the in-cell ELISA is an accurate method to determine relative protein levels and degree of post-translational modification (PTM) among various cell types. The In-cell ELISA may be performed in 96- or 384-well microplates, are scalable and conserve cell culture and treatment reagents. Because assay data are measured using a standard ELISA plate reader or infrared imaging system, the results are readily available for analysis. Also, the assay is amenable to automation, which is ideal for siRNA studies and drug screens.

About the Thermo Scientific In-Cell ELISA Kits

The In-Cell ELISA Near IR Kit enables simultaneous detection of two different targets within the same well (e.g., PTM- and unmodified-forms of a protein, or two entirely different proteins). Simultaneously detecting targets within the same microplate well eliminates variability caused by differences in cell plating. The expression levels of the protein(s) are monitored using primary antibodies specific to the targets and corresponding species-specific Near Infrared DyLight Fluor-Conjugated Secondary Antibodies. Relative protein activation or inactivation is determined as a ratio of modified protein levels to the corresponding unmodified protein measured using a modification-specific antibody and an antibody to unmodified protein, respectively.

The In-Cell ELISA Colorimetric Detection Kit enables the levels of target proteins to be compared in different wells. The relative amount of protein is determined using target-specific primary antibodies and a horseradish peroxidase (HRP)-conjugated detection reagent. Following the colorimetric measurement of HRP activity, the provided whole cell stain, Janus Green, is used to determine cell number. After staining, the results are analyzed by normalizing the absorbance (HRP activity) values to cell number, which adjusts for the cell plating differences.

Highlights of the In-Cell ELISA Kits:

• Two detection formats available
  • Colorimetric – compatible with standard ELISA plate readers
  • Near Infrared – compatible with the LI-COR Odyssey Infrared Imaging System
• Simple, fast assay format
• Ability to perform multiplex experiments
• No special sample preparation required
• Higher throughput compared with Western blotting
• Cost efficient (one well vs. one lane) compared to Western blotting

Applications for In-Cell ELISA Kits:

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

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. 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.

Multiplex analysis of targets in multiple cell lines using the In-Cell ELISA Colorimetric Detection Kit. A594, A431 and HeLa cells were treated with or without 100ng/ml EGF and then the levels of ppEGFR, ppERK and tubulin were measured using the In-Cell ELISA Colorimetric Detection Kit. Results demonstrate that EGF increases the levels of ppEGFR and ppEGF.

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 100ng/ml EGF or both 100ng/ml EGF and 25 nM PD168393 (panel A). Graphing the data shows inhibition but not complete blocking of phosphorylation by 25nM PD168393 (panel B).

Related Products and Links:
Target-Specific In-Cell ELISA Kits – more information on how these optimized kits can be used
ELISA reagent reservoirs and sealing tape
Secondary Antibodies and Conjugates For ELISAs and Western Blotting
DyLight Fluor Conjugated Secondary Antibodies and Biotin Binding Proteins