In-cell ELISA protocol
Licia Miller Product Manager
In-cell ELISA (also known as cell-based ELISA, cell western blot, or cytoblot) is a widely accepted immunocytochemistry assay that uses a colorimetric or fluorescent readout to quantify a target protein or post-translational modifications of a target protein in cultured cells.
Here we provide a general protocol for 96-well microplates. The general process includes: culturing cells (treated as needed) and seeding them into coated 96-well microplates; after fixation and permeabilization, adding primary antibodies to the wells and incubating, then adding labeled secondary antibodies, and finally detection and analysis.
1. Prepare cells
Adherent cells can be grown in the recommended assay microplates or seeded directly into assay microplates and allowed to attach for several hours or overnight.
Materials required:
- 96-well microplates: clear bottom (black walls required for Fluorescent antibodies), preferably coated with amine/collagen etc. for optimal cell culture conditions
-Multichannel pipette (recommended)
- Distilled or deionized water
- 10× phosphate buffered saline ( PBS ) (80 mM Na2HPO4, 14 mM KH2PO4, 1.4 M NaCl, 27 mM KCl, pH adjusted to 7.4 with NaOH)
- 1× PBS: Prepare at the beginning of the experiment. Dilute 45 mL of 10× PBS in 405 mL of distilled or deionized water and mix thoroughly. Store at room temperature.
- 400× Tween-20 (20% aqueous solution)
- 1× Wash Buffer: Prepare at the beginning of the experiment. Dilute 625 µL 400× Tween-20 in 250 mL 1× PBS and mix well. Store at room temperature.
Experimental procedures
1. Seed the cells at the desired density in a 96-well microplate. For a 384-well microplate, seed at one-fourth the density of a 96-well plate .
Notes: The optimal cell seeding density depends on the cell type and should be determined for each experiment. For example, in a 96-well microplate, 25,000 - 50,000 HeLa cells should be seeded per well.
2. Allow cells to adhere for several hours or overnight.
3. Treat attached cells as needed , using a total volume of 100 µL for 96-well plates (1/4 the volume for 384-well plates) . Up to 10% serum is acceptable in the culture medium.
Notes: You should determine the duration of cell treatment. When treating cells with a drug of interest, we recommend including both untreated cells and cells treated with the drug solvent.
2. Fix cells to microplate
Materials required:
- 10× Phosphate Buffered Saline (PBS): 80 mM Na2HPO4, 14 mM KH2PO4, 1.4 M NaCl, 27 mM KCl, pH adjusted to 7.4 with NaOH.
- 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 of 10× PBS and 5.0 mL of 20% paraformaldehyde. CAUTION: Paraformaldehyde is toxic and should be prepared and used in a fume hood. Dispose of paraformaldehyde according to local regulations.
- 1× PBS prepared at the beginning of the experiment
- 96-well microplates. Clear bottom (black walls required for Fluorescent antibodies), preferably coated (amine/collagen) for optimal cell culture
-Multichannel pipette (recommended)
Experimental procedures
1. Immediately add an equal volume (100 μL) of 8% paraformaldehyde solution to the wells containing culture medium . Incubate for 15 minutes.
Warning: Paraformaldehyde is toxic and should be prepared and used in a fume hood. Dispose of paraformaldehyde according to local regulations.
2. Gently aspirate the paraformaldehyde solution in the microplate and wash the microplate three times with 300 μL 1× PBS per well.
3. Add 100 μL of 1× PBS to the wells . Cover the microplate with a sealer.
Tips: The microplate can now be stored at 4°C for several days. For long-term storage, 0.02% sodium azide can be added to PBS.
Warnings: Paraformaldehyde and sodium azide are toxic and should be handled with care and disposed of according to local regulations.
Notes: The microplate should not be allowed to dry out at any time during or prior to the assay.
3. Cell Permeabilization
It is recommended to use a plate shaker (~300 rpm) during incubation steps. After any step involving removal of buffers or solutions, the plate should be gently tapped on a paper towel to remove all solution from the wells.
Materials required:
- 100× Triton X-100 (10% aqueous solution)
- 1× PBS
- 1× Permeabilization Buffer: Prepare immediately before use. Dilute 250 µL Triton X-100 in 24.75 mL 1× PBS and mix.
- 10× Blocking Buffer
- 2× Blocking Buffer: Prepare immediately before use. Dilute 5 mL 10× Blocking Buffer in 20 mL 1× PBS.
-Multichannel pipette (recommended)
- Flatbed shaker
Experimental procedures
1. Remove PBS and add 200 μL of freshly prepared 1× Permeabilization Buffer to each well .
2. Incubate for 30 minutes .
Tips: It is recommended to use a plate shaker (~300 rpm) during the incubation step.
Any step involving the removal of buffers or solutions should be followed by tapping the plate gently on a paper towel to remove all solution from the wells.
3. Remove 1× Permeabilization Buffer and add 200 μL of 2× Blocking Solution to each well.
4. Incubate for 2 hours.
Notes: It is recommended to use a plate shaker (~300 rpm) during the incubation step.
Any step involving the removal of buffers or solutions should be followed by tapping the plate gently on a paper towel to remove all solution from the wells.
4. Primary Antibody Incubation
We must omit the primary antibody in at least one well to determine the background signal of the experiment, which can be subtracted from all measured data. This should be done for each experimental condition and each detection antibody.
Materials required:
- Primary antibodies identified by intracellular ELISA
- 10× Blocking Buffer
- 1 x PBS
- 1× Incubation Buffer: Prepare immediately before use. Dilute 2.5 mL 10× Blocking Buffer in 22.5 mL 1× PBS.
-Multichannel pipette (recommended)
Experimental procedures
1. Dilute the provided antibody stock solution in 1× incubation buffer to prepare a 1× primary antibody solution.
2. Remove 2× blocking solution, add 100 µL of diluted primary antibody solution to each well, and incubate overnight at 4℃.
Tips: To determine background signal, omit the primary antibody from at least one well containing cells for each experimental condition and detection antibody used.
5. Secondary Antibody Incubation
Materials required:
- 1× Wash Buffer prepared at the beginning of the experiment
- Appropriate Fluorescent- or HRP-labeled secondary antibodies
- 10× Blocking Buffer
- 1× PBS
- 1× Incubation Buffer: Prepare immediately before use. Dilute 2.5 mL 10× Blocking Buffer in 22.5 mL 1× PBS.
-Multichannel pipette (recommended)
Experimental procedures
1. Prepare 1× secondary antibody solution by diluting in 1× Incubation Buffer as directed in the kit instructions (or antibody datasheet) .
Notes: Protect fluorescent-labeled antibodies from light.
2.wash the microplate three times with 250 μL 1× wash buffer / well.
3. Remove 1× wash buffer, add 100 µL 1× secondary antibody solution to each well and incubate for 2 hours .
4. Discard the secondary antibody solution and wash 4 times with 250 µL 1× wash buffer/ well.
Notes: Do not remove the final wash buffer.
6. Measurement Signal
Materials required:
- 70% ethanol
- Appropriate microplate reader: For HRP detection, use a microplate reader capable of measuring absorbance at 650 nm (preferably in kinetic mode) or 450 nm. For fluorescent-labeled antibodies, use the appropriate imaging system (such as LI-COR® Odyssey® or Aerius® near-infrared imaging system)- For HRP detection, HRP substrate solution
- 0.5 M hydrochloric acid
Experimental procedures
1. If using a fluorescent-conjugated secondary antibodies, wipe the bottom of the microplate and the scanner surface with 70% ethanol before scanning the microplate on the LI-COR® Odyssey® System .
Tips: Use the 700 nm and 800 nm channels according to the manufacturer's instructions (recommended intensity range 5-7).
2. For HRP -conjugated secondary antibodies, remove the last wash and wipe the microplate face down to remove excess liquid . Then add 100 µL of HRP substrate.
Pop any bubbles , set the microplate reader parameters as follows, and record the blue color change:
- Mode: Kinetic
- Wavelength: 650nm
- Duration: 30 minutes
- Interval: 20 seconds to 1 minute
- Shake: Shake between readings
- Alternatives: Instead of a kinetic readout, at a user-defined time, record the endpoint OD at 640 nm, or stop the reaction by adding 100 µL of 0.5M HCl and record the OD at 450 nm.
3. Save the data and export the raw data to Excel.
Tips: Analyze data using LI-COR® ICW Software or other appropriate data analysis software (e.g., Microsoft Excel or GraphPad Prism).
Tips and tricks for intracellular ELISA :
1. About cells
-Cell seeding density, culture medium, and other growth conditions are critical for successful and reproducible experiments.
- User should define cell type specific parameters.
2. Cell Attachment and Fixation
-Adherent cells can be grown and processed directly in assay microplates.
- Cell attachment can be checked microscopically.
-When cells are fully attached, the medium can be removed and replaced with medium containing treatment reagents.
- Medium containing up to 10% fetal serum will not interfere with cell fixation and crosslinking to the microplate but may interfere with processing reagents.
3. Cell Treatment
- When using drugs (inhibitors or activators), experiments should include both untreated cells and cells treated with only the drug solvent.
4. Cell Seeding Density
- The cell seeding density in the assay microplates depends on the cell type. It depends on the cell size (diameter for adherent cells) and the abundance of the target protein.
-Using a single layer is generally recommended to obtain a robust signal.
- The number of cells to be seeded can be determined by observing the cell density of serial dilutions under a microscope.
- For adherent cells, prepare serial dilutions of cells in a microplate (with similar well sizes) and observe the cell density under a microscope. Within the appropriate cell density range, higher cell densities will produce stronger overall signals and increase the detection of small signals . For example, HeLa and HepG2 cells should be seeded at 25,000 to 50,000 cells per well, while human fibroblasts ( HdFN ) - 15,000 to 25,000 cells per well.
5. Controls
Omit the primary antibody in at least one well to provide a background signal for the experiment that can be subtracted from all measurements. This should be done for each experimental condition and each detection antibody.
6. Scalability
The assay can also be performed in a 384-well microplate format using one-quarter the volume of reagents and cells.
For more product details, please visit Aladdin Scientific website.