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In-cell ELISA protocol

In-Cell ELISA (also known as cell-based ELISA, in cell western or cytoblot) is an immunocytochemistry method used to quantify target protein or post-translational modifications of the target protein,

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Last edited Thu 09 Mar 2023

​​​Here we provide a general protocol for a 96-well microplate that can be used for all our In-Cell ELISA kits as well as with our antibodies characterized for In-Cell ELISA. 

Cells are cultured (and treated if required) and seeded into a coated 96-well microplate. After fixation and permeabilization, a primary antibody is added to the well and is incubated, followed by the addition of a labeled secondary antibody. Detection can be colorimetric or fluorescent for a single target using our In-Cell ELISA kits or primary antibodies characterized for In-Cell ELISA. Dual targets can be quantified using IR-conjugated secondary antibodies in our In-Cell ELISA kits. Kits include highly-specific, well-characterized primary antibodies generated from mouse or rabbit, and appropriately conjugated secondary antibodies for colorimetric, fluorescent or infra-red detection.  ​​

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

1

Seed cells into 96-well microplate at desired density.

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

2

Allow cells to adhere for several hours or overnight

3

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

1

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

2

Incubate for 15 min

3

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

4

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)
  • 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.
  • 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

1

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

2

Incubate for 30 min

3

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

4

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

1

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

2

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

1

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

2

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

3

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

Incubate for 2 hours.

4

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

1

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).

2

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.

3

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 required

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

Steps

1

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.
2

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

3

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

4

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

1

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

2

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

3

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