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Colorimetric Cell-Based ELISA Kit Protocol

Colorimetric Cell-Based (In-Cell) ELISA Protocol | Assay Genie
Assay Genie · Cell-Based ELISA

Colorimetric cell-based ELISA protocol

Measure total or phosphorylated target proteins directly in fixed cells. This sample protocol covers buffer preparation, experimental design, the assay procedure and data normalisation.

Jump to protocol
Buffer prep16-step assayData normalisation
96-well
Plate format
450 nm
Read wavelength
GAPDH
Normalisation
Duplicate
Or triplicate

Overview

Below are the steps for a colorimetric cell-based ELISA kit - buffer preparation, experimental design and the assay protocol. Allow all solutions to reach room temperature before use, and read the whole manual before starting.

Buffer preparation

ReagentPreparation
10x TBS1x TBS washes cells on the plate. Prepare by adding 1 volume 10x TBS to 9 volumes ddH2O.
Fixing SolutionNot provided. Fixes cells after culture: add formaldehyde to 1x TBS with light mixing. Use 4% formaldehyde for adherent cells and 8% for suspension/loosely attached cells (37% formaldehyde, e.g. Sigma F-8775).
Quenching BufferReady-to-use. Inactivates endogenous peroxidase activity in the seeded cells.
Blocking BufferReady-to-use. Blocks additional binding sites in each well.
1x Wash BufferProvided as 15x. Prepare by adding 1 volume 15x Wash Buffer to 14 volumes ddH2O.
100x Anti-Phospho Target Primary Antibody100x solution; dilute 1:100 in Primary Antibody Diluent. Diluted antibody stores at 4°C for up to two weeks. (Phospho kits only.)
100x Anti-Target Primary Antibody100x solution; dilute 1:100 in Primary Antibody Diluent. Stores at 4°C up to two weeks.
100x Anti-GAPDH Primary AntibodyMouse monoclonal, specific for GAPDH. 100x; dilute 1:100 in Primary Antibody Diluent. Used as internal positive control for normalisation.
HRP-Conjugated Anti-Rabbit IgGReady-to-use secondary antibody to detect target-bound primary rabbit antibodies.
HRP-Conjugated Anti-Mouse IgGReady-to-use secondary antibody to detect target-bound primary mouse antibodies (e.g. anti-GAPDH).
Primary Antibody DiluentReady-to-use. Used to dilute the provided antibodies.
Ready-to-Use SubstrateWarm to room temperature before use; light-sensitive - keep in the dark.
Stop SolutionReady-to-use; contains 2 N sulfuric acid and is corrosive. Wear eye protection and gloves.
Crystal Violet SolutionReady-to-use intense stain for cell nuclei; avoid contact with skin and clothing.
SDS SolutionReady-to-use; solubilises Crystal Violet for staining. Store at room temperature.

Additional materials required

These are not provided in the kit but are needed to run the assay.

Materials & equipment (not provided)
Microplate reader measuring absorbance at 450 nm (and 595 nm for optional Crystal Violet staining)
Micropipettes covering 1 µL to 1 mL
37% formaldehyde (e.g. Sigma F-8775) or equivalent
Deionised or sterile water
Squirt bottle; manifold dispenser
Multi-channel pipette reservoir or automated microplate washer
Graph paper or software for logarithmic plots
Absorbent paper or vacuum aspirator
Test/microfuge tubes (>=1 mL)
Orbital shaker
Poly-L-Lysine (e.g. Sigma P4832) for suspension cells

Experimental design

ConsiderationDetail
Cell lineMust express the target protein. Adherent cells work directly; for suspension/loosely attached cells, first coat wells with 100 µL of 10 µg/mL Poly-L-Lysine for 30 min at 37°C and fix with 8% formaldehyde.
Cell number & sensitivityCells should be ~75-90% confluent. For HeLa, seed ~30,000 cells/well overnight before treatment.
Cell treatmentTreat with inhibitors, activators or stimulators (chemicals, proteins/peptides), UV or serum starvation as required.
Positive & negative controlsUse Anti-GAPDH (included) as the internal positive control for normalisation. Negative controls are the HRP-conjugated secondaries alone (no primary). Run controls on the same plate as the target.
Accuracy & precisionRun each condition in duplicate or triplicate.

Protocol

The 16-step colorimetric cell-based ELISA assay procedure.

1
Seed 200 µL of 20,000 adherent cells in culture medium per well of the (sterile, culture-treated) 96-well plate. For suspension/loosely attached cells, first coat wells with 100 µL of 10 µg/mL Poly-L-Lysine for 30 min at 37°C.
2
Incubate cells overnight at 37°C, 5% CO2.
3
Treat the cells as desired.
4
Remove culture medium and rinse twice with 200 µL 1x TBS.
5
Fix cells with 100 µL Fixing Solution for 20 min at room temperature (4% formaldehyde for adherent, 8% for suspension). Seal plates with Parafilm; wear appropriate PPE as the solution is volatile.
6
Remove Fixing Solution and wash 3x with 200 µL 1x Wash Buffer for 5 min each with gentle shaking. The plate can be stored at 4°C for a week. Tap gently on absorbent paper to remove solution completely.
7
Add 100 µL Quenching Buffer; incubate 20 min at room temperature.
8
Wash 3x with 1x Wash Buffer, 5 min each, with gentle shaking.
9
Add 200 µL Blocking Buffer; incubate 1 hour at room temperature.
10
Wash 3x with 200 µL 1x Wash Buffer, 5 min each, with gentle shaking.
11
Add 50 µL of each 1x Primary Antibody to the corresponding wells; cover with Parafilm and incubate 16 hours (overnight) at 4°C. If target expression is high, 2 hours at room temperature with gentle shaking may suffice.
12
Wash 3x with 200 µL 1x Wash Buffer, 5 min each, with gentle shaking.
13
Add 50 µL of the appropriate 1x Secondary Antibody and incubate 1.5 hours at room temperature with gentle shaking. Use anti-Rabbit IgG-HRP for the target primary wells and anti-Mouse IgG-HRP for the anti-GAPDH wells.
14
Wash 3x with 200 µL 1x Wash Buffer, 5 min each, with gentle shaking.
15
Add 50 µL Ready-to-Use Substrate per well; incubate 30 min at room temperature in the dark with gentle shaking (substrate is light-sensitive).
16
Add 50 µL Stop Solution per well and read OD at 450 nm immediately on the microplate reader.

Optional: Crystal Violet cell staining

Crystal Violet binds cell nuclei, giving absorbance at 595 nm proportional to cell number - useful for normalisation.

1
After reading at 450 nm, wash the plate twice with 200 µL Wash Buffer and twice with 200 µL 1x TBS (5 min each). Tap dry and air-dry 5 min at room temperature.
2
Add 50 µL Crystal Violet Solution per well; incubate 30 min at room temperature on the shaker. (Intense stain - avoid skin and clothing contact.)
3
Tip off the Crystal Violet, then rinse wells in running water/ddH2O until no more colour comes off. Dry ~30 min.
4
Add 100 µL SDS Solution per well; incubate on the shaker at room temperature for 1 hour.
5
Read absorbance at 595 nm. If too high, dilute the solubilised Crystal Violet 10x with ddH2O on a separate plate.

Data normalisation

MethodHow
GAPDH normalisationNormalise the target OD450 values using the OD450 values obtained for GAPDH.
Crystal Violet normalisationNormalise OD450 readings using OD595 values via the ratio OD450 / OD595.

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FAQs

What is a cell-based (in-cell) ELISA?

It measures total or phosphorylated target proteins directly in cells fixed in the wells of a 96-well plate, using primary and HRP-conjugated secondary antibodies with colorimetric detection at 450 nm.

How do I normalise the data?

Normalise target OD450 to the internal GAPDH control (OD450), or use optional Crystal Violet staining and take the OD450/OD595 ratio to correct for cell number.

Can I use suspension cells?

Yes. Coat the wells with 10 µg/mL Poly-L-Lysine for 30 min at 37°C before seeding, and fix with 8% formaldehyde instead of 4%.

What controls should I run?

Include anti-GAPDH as the internal positive/normalisation control and the HRP-conjugated secondaries alone (no primary) as negative controls, on the same plate, in duplicate or triplicate.

Running a cell-based ELISA?

Our scientific team can help with kit selection, controls and normalisation for your target.

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