In-cell ELISA protocol

In-Cell ELISA is a powerful, high-throughput immunocytochemistry technique used to quantify protein expression and post-translational modifications directly in cultured cells. This method, also known as cell-based ELISA or in-cell western, enables researchers to analyze multiple samples simultaneously in a 96-well format. Abcam’s In-Cell ELISA kits streamline the process with optimized antibodies and detection reagents for colorimetric, fluorescent, or infrared readouts. The protocol is ideal for drug screening, pathway analysis, and biomarker validation. With minimal sample handling and no need for cell lysis, In-Cell ELISA eliminates the requirement for preparing cell lysate, which is often necessary in traditional ELISA or western blot assays. In-Cell ELISA is one of several assays used for protein quantification, offering a sensitive and reproducible alternative to traditional Western blotting, making it a preferred choice for many cell biology and pharmacology labs.

Introduction

The In-Cell ELISA protocol from Abcam provides a robust method for quantifying intracellular proteins in fixed cells. Unlike traditional ELISA, which requires cell lysis, this technique preserves cellular architecture, allowing for spatial and quantitative analysis of protein expression. It is particularly useful for detecting changes in protein levels or modifications in response to treatments. The protocol is compatible with a wide range of cell types and is adaptable for both adherent and suspension cells. Abcam’s kits include validated antibodies and detection systems, including detection antibodies for identifying the specific protein of interest, ensuring high specificity and reproducibility. The protocol utilizes primary and secondary antibodies to ensure sensitive and specific detection of the target analyte. This makes In-Cell ELISA a valuable tool for researchers studying signaling pathways, drug responses, and cellular phenotypes.

Background and principles

In-Cell ELISA is a high-throughput, cell-based immunoassay that combines the core principles of ELISA and immunocytochemistry to quantify protein expression directly in fixed cells. Cells are seeded into a coated 96-well microplate, treated as required, and then fixed to preserve protein localization. After permeabilization, a target-specific primary antibody is added, followed by a labeled secondary antibody. Detection can be colorimetric or fluorescent for single-target analysis using our In-Cell ELISA kits or primary antibodies characterized for In-Cell ELISA. For dual-target detection, IR-conjugated secondary antibodies in our In-Cell ELISA kits enable multiplexed analysis. These kits include highly specific mouse or rabbit primary antibodies and secondary antibodies optimized for colorimetric, fluorescent, or infrared detection. In sandwich ELISA formats, two primary antibodies may be used, one for capture and one for detection, each recognizing distinct epitopes. Signal detection is achieved via enzyme- or fluorophore-conjugated secondary antibodies, with output directly proportional to the target protein level. This method supports normalization to cell number or housekeeping proteins and is ideal for pathway analysis, drug screening, and detecting subtle changes in protein expression or phosphorylation.

Comparison to other methods

Hints and tips for a successful In-Cell ELISA

• Cells

  • Cell seeding density, culture medium, and other growth conditions are key to a successful and reproducible experiment.
  • Cell-type-specific parameters should be defined by the user.

• Cell attachment and fixation

  • Adherent cells can be grown and treated directly in the assay microplate.
  • Cell attachment can be checked with a microscope.
  • When the cells are fully attached, the media can be removed and replaced with media containing the treatment reagent.
  • Culture media containing up to 10% fetal serum does not interfere with cell fixation and cross-linking to the microplate but may interfere with the treatment reagent.

• Cell treatment

  • When using a drug (inhibitor or activator), we should include in the experiment both untreated cells and cells treated with only the drug solvent.

• Cell seeding density:

  • The cell seeding density of the assay microplate is dependent on the cell type. It depends on the cell size (diameter, in case of the adherent cells) and the abundance of the target protein.
  • As a general guideline, a monolayer is recommended for robust signal.
  • The cell seeding can be determined experimentally by microscopic observation of the cell density of serially diluted cells.
  • For adherent cells, prepare a serial dilution of the cells in a microplate (of similar well dimensions) and observe the cell density under a microscope. Working on the high end of this range will generate a stronger overall signal and allow the detection of small signal increases. For example, HeLa and HepG2 cells should be seeded from 25,000 to 50,000 cells per well, while human fibroblasts (HdFN) - from 15,000 to 25,000 cells per well.

• Controls

  • Omit primary antibody in at least one well to provide a background signal for the experiment, which can be subtracted from all measured data. This should be done for each experimental condition and with each detector antibody.

• Scalability

  • This assay can also be performed in 384-well microplate format by using ¼ of the volume of reagents and cells, as specified in the below protocol.

Stage 1 - Prepare cells

Adherent cells can be grown in the recommended assay microplates or seeded directly into the assay microplate and allowed to attach for several hours or overnight.

Materials required

• ​​​​​96-well microplate: clear bottom (black wall necessary for IRDye® only), preferably coated (amine/collagen) for optimal cell culture
• Multichannel pipette (recommended)
• Distilled or deionized water
• 1X PBS
• Wash buffer (for example ab206977)

To facilitate the in-step ELISA process, consider using an In-Cell ELISA Support Pack with (ab111542) or without plates (ab111541).

Steps

Seed cells into 96-well microplate at desired density.

For a 384-well microplate, seed at ¼ of the density.

Allow cells to adhere for several hours or overnight

Treat the attached cells as desired in total volume of 100 µL media for 96-well microplate (or ¼ of volume of 384-well microplate)

Up to 10% serum is acceptable in the media.

Stage 2 - Fix cells to microplate

Materials required

• 20% paraformaldehyde
• Distilled or deionized water
• 8% paraformaldehyde: prepare immediately before use. Mix 6.25 mL of distilled or deionized water with 1.25 mL 10X PBS and 5.0 mL 20% paraformaldehyde.  Note: Paraformaldehyde is toxic and should be prepared and used in a fume hood. Dispose of paraformaldehyde according to local regulations.
• 1X PBS
• 96-well microplate.  Clear bottom (black wall necessary for IRDye® only), preferably coated (amine/collagen) for optimal cell culture
• Multichannel pipette (recommended)

Steps

Immediately add an equal volume (100μL) of 8% paraformaldehyde solution to the wells containing culture media

Incubate for 15 min

Gently aspirate the paraformaldehyde solution from the microplate and wash the microplate 3 times with 300 μL 1X PBS per well

Add 100 μL 1X PBS to the wells.

• Cover the microplate with the provided seal.
• The microplate can now be stored at 4°​C for several days.
• For prolonged storage, supplement PBS with 0.02% sodium azide.

Stage 3 - Permeabilize cells

It is recommended to use a plate shaker (~300 rpm) during incubation steps. Any step involving removal of buffer or solution should be followed by gently tapping the plate on a paper towel to remove all solutions from the wells.

Materials required

• 100X Triton X-100 (10% solution in H2O; NP-40 can also be used)
• 1X PBS
• 1X Permeabilization Buffer: prepare immediately before use. Dilute 250 µL of Triton X-100 in 24.75 mL 1X PBS and mix well (NP-40 can also be used).
• 10X blocking buffer
• 2X Blocking Solution: prepare immediately before use. Dilute 5 mL 10X blocking solution in 20 mL 1X PBS.
• Multichannel pipette (recommended)
• Plate shaker

Steps

Remove PBS and add 200 μL freshly prepared 1X permeabilization buffer to each well

Incubate for 30 min

Remove 1X permeabilization buffer and add 200 μL of 2X blocking solution to each well

Incubate for 2 hours

Stage 4 - Incubate with primary antibody

We must omit the primary antibody in at least one well to determine a background signal for the experiment, which can be subtracted from all measured data. This should be done for each experimental condition and with each detector antibody.

Materials required

• ​​​​​In-Cell ELISA characterized primary antibody
  • Our primary antibodies are supplied with a recommended final concentration for In-Cell ELISA, which can be found on each antibody datasheet.
• 10X blocking buffer
• 1X PBS
• 1X incubation buffer: prepare immediately before use. Dilute 2.5 mL of 10X blocking solution in 22.5 mL 1X PBS.
• Multichannel pipette (recommended)

Steps

Prepare 1X primary antibody solution by diluting the provided stock antibody in 1X incubation buffer

Remove 2X blocking solution and add 100 µL of the diluted primary antibody solution to each well

Incubate overnight at 4°C.

Stage 5 - Incubate with the secondary antibody

Materials required

• Wash buffer (for example ab206977)
• Appropriate IRDye®, AP-labeled,​ or HRP-labeled secondary antibody
• 10X blocking solution
• 1X PBS
• 1X incubation buffer: prepare immediately before use. Dilute 2.5 mL of 10X blocking solution in 22.5 mL 1X PBS.
• Multichannel pipette (recommended)

Steps

Remove primary antibody solution and wash the microplate 3 times with 250 μL 1X wash buffer per well

Prepare 1X secondary antibody solution by diluting as directed in the kit protocol (or antibody datasheet) in 1X incubation buffer

Remove 1X wash buffer and add 100 µL 1X secondary antibody solution to each well

Incubate for 2 hours.

Remove secondary antibody solution and wash 4 times with 250 µL 1X wash buffer per well

Stage 6 - Measure signal

Materials required

• 70% ethanol
• Plate reader
• For HRP detection, HRP substrate solution
• 0.5 M HCl

Steps

If using dual detection kits (with IRDye®​ conjugated secondary antibodies), wipe the bottom of the microplate and the scanner surface with 70% ethanol and scan the microplate on the LI-COR® Odyssey® system

Use both 700 nm and 800 nm channels according to the manufacturer's instructions (the suggested intensity range is 5-7).

For HRP conjugated secondary antibodies, remove the last wash and blot the microplate face down to remove excess liquid

• Add HRP or AP substrate.

• Set up your plate reader to observe the color change or fluorescence at the expected wavelength.

• Pop any bubbles and immediately record the blue color development in the microplate reader prepared as follows:

  • Mode: Kinetic
  • Time: 30 min
  • Interval: 20 sec–1 min
  • Shaking: Shake between readings
  • Alternative: In place of kinetic reading, at a user-defined time, record the endpoint OD, or stop the reaction by adding 100 µL 0.5M HCl and record the OD.

Note: If you stop the reaction before measuring the endpoint OD,  the wavelength to use will likely be lower.

Save the data and export raw data to Excel

Stage 7 - Whole cell staining with Janus Green (optional)

The signal of antibody-specific complexes can be normalized to the intensity of Janus Green staining to account for differences in cell seeding density. A plate shaker (~300 rpm) is recommended during all incubation steps.

Materials require

• 0.3% solution Janus Green Stain (ab111622) in water
• Distilled or deionized water
• 0.5 M HCl
• Plate reader
• Multichannel pipette (recommended)

Steps

Incubate your cells with Janus Green Stain

• Empty microplate wells and add 50 µL of 1X Janus Green Stain per well.
• Incubate the microplate for 5 min at room temperature.

Remove dye and wash the microplate 5 times in deionized water or until excess dye is removed

Remove last water wash, blot to dry, add 200 µL of 0.5 M HCl and incubate for 10 min.

Measure using a LI-COR® Odyssey®​ scanner in the 700 nm channel.

• Alternatively, measure OD at 595 nm in a standard microplate spectrophotometer.

Stage 8 - Data analysis

Steps

Correct the raw in-cell ELISA signal for the background by subtracting the mean signal of well(s) incubated in the absence of the primary antibody from all other readings

Optional: correct the Janus Green signal of wells that do not contain cells from all other Janus Green readings.

Normalize the in-cell ELISA signal by dividing the background-corrected in-cell ELISA signal by the "background-corrected" Janus Green signal.

Applications

In-Cell ELISA is widely used in drug discovery, signal transduction studies, and biomarker validation. It enables researchers to monitor protein expression, phosphorylation, and other post-translational modifications in response to various treatments. The technique is ideal for high-throughput screening of inhibitors or activators, time-course experiments, and dose-response analyses. It is also valuable in cancer research, neuroscience, and immunology, where quantifying protein dynamics in situ is critical. Abcam’s In-Cell ELISA kits support multiplexing, allowing simultaneous detection of multiple targets, and are compatible with automated plate readers, enhancing reproducibility and scalability.

Limitations

While In-Cell ELISA offers many advantages, it has some limitations. It requires cell fixation, which precludes live-cell analysis and may affect epitope accessibility. The technique is less suitable for proteins with low expression levels or those not well-retained during fixation. Signal intensity can be influenced by cell density and fixation conditions, necessitating careful optimization. Multiplexing is limited by the availability of compatible antibodies and detection systems. Additionally, the method provides average signal per well, lacking single-cell resolution. Despite these constraints, In-Cell ELISA remains a powerful tool for many applications when properly optimized.

Troubleshooting

Common issues in In-Cell ELISA include weak or inconsistent signals, high background, and poor reproducibility. To address low signal, ensure optimal cell seeding density and verify antibody specificity and concentration. High background may result from insufficient washing or non-specific antibody binding; thus, you should optimize blocking conditions and antibody dilutions. Uneven staining can stem from inconsistent cell attachment; use coated plates and monitor cell confluency. If signal variability persists, standardize fixation and permeabilization steps. Always include appropriate controls, such as untreated cells and isotype antibodies. Abcam provides detailed troubleshooting tips and technical support to help optimize your assay for reliable results.