Colorimetric Cell-Based ELISA Kit Protocol

10x TBS

1x TBS is used to wash cells seeded on the plate. 1x TBS can be prepared by adding 1 volume of 10x TBS provided in the kit to 9 volumes of ddH2O.

Fixing Solution

This solution is NOT provided. Fixing Solution is used to fix cells after cell culture. It is prepared by adding formaldehyde to 1x TBS with light mixing. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells. 37% formaldehyde can be purchased from Sigma Cat# F-8775.

Quenching Buffer

This solution is provided as ready-to-use. Quenching Buffer is used to inactivate the endogenous peroxidase activity of the seeded cells.

Blocking Buffer

This solution is provided as ready-to-use. Blocking Buffer is used to block additional binding sites in each well.

1x Wash Buffer

This buffer is provided as a 15x solution. 1x Wash Buffer can be prepared by adding 1 volume of 15x Wash Buffer provided in the kit to 14 volumes of ddH2O.

100x Anti-Phospho Target Primary Antibody

The supplied antibody is a 100x solution. Make 1:100 dilutions in Primary Antibody Diluent prior to use. The diluted primary antibody can be stored at 4°C for up to two weeks. (*Cell-Based Phospho ELISA Kits only)

100x Anti-Target Primary Antibody

The supplied antibody is a 100x solution. Make 1:100 dilutions in Primary Antibody Diluent prior to use. The diluted primary antibody can be stored at 4°C for up to two weeks.

100x Anti-GAPDH Primary Antibody

This antibody is a mouse monoclonal antibody. This antibody was tested to be specific for GAPDH. The supplied antibody is a 100x solution. Make 1:100 dilutions in Primary Antibody Diluent prior to use. The diluted primary antibody can be stored at 4°C for up to two weeks.This antibody is a mouse monoclonal antibody. This antibody was tested to be specific for GAPDH. The supplied antibody is a 100x solution. Make 1:100 dilutions in Primary Antibody Diluent prior to use. The diluted primary antibody can be stored at 4°C for up to two weeks.

HRP-Conjugated Secondary Anti-Rabbit IgG Antibody

This solution is provided as ready-to-use. HRP-Conjugated Anti-Rabbit IgG Antibody is used as the secondary antibody to detect the target-bound, primary rabbit antibodies.

HRP-Conjugated Secondary Anti-Mouse IgG Antibody

This solution is provided as ready-to-use. HRP-Conjugated Anti-Mouse IgG Antibody is used as the secondary antibody to detect the target-bound, primary mouse antibodies.

Primary Antibody Diluent

This solution is provided as ready-to-use. Use this solution to dilute the provided antibodies.

Ready-to-Use Substrate

This solution is provided as ready-to-use. Ready-to-Use Substrate must be warmed to room temperature before use. Keep away from light as this solution is light-sensitive.

Stop Solution

This solution is provided as ready-to-use. Stop Solution must be handled with caution as it contains 2 N Sulfuric Acid (H2SO4) and is corrosive. Wear eye protection and gloves when handling.

Crystal Violet Solution

This solution is provided as ready-to-use. Crystal Violet is an intense stain used to stain cell nuclei. Avoid contact with skin and clothing.

SDS Solution

This solution is provided as ready-to-use. SDS is used to solubilize the Crystal Violet in preparation for cell staining. Store this solution at room temperature or warm up to room temperature prior to use if stored at 4°C.

Adhesive Plate Sealers

Provided for long term storage of plate if necessary.

1.

Cell Line: The cell line must express the target protein. This protocol can be used directly for adherent cells. For suspension cells and loosely attached cells, two steps are required: 1) Coat the plates with 100 µl of 10 µg/ml Poly-L-Lysine (Sigma Cat# P4832, not included) to each well of the 96-well plate for 30 minutes at 37°C before proceeding to Step 1 of Assay Protocol (on page 16). Use 8% formaldehyde to fix the cells on Step 5 of Assay Protocol.

2.

Cell Number and Sensitivity: The number of cells plated onto the 96-well plates depends on the expression level of target protein in the cells, cell size, treatment conditions and incubation time. The cells used for testing should be around 75-90% confluent. Typically for HeLa cells, seed 30,000 cells per well overnight for treatment the following day.

3.

Cell Treatment: The cells can be treated with inhibitors, activators, stimulators (ie. chemicals, proteins/peptides) or a combination of the substances listed above. The cells can be treated with UV and serum starvation to meet the needs of the end-user.

4.

Positive and Negative Controls: Mouse Anti-GAPDH Antibody (included) should be used to detect the internal positive controls for normalization of OD values of the target protein. The negative controls are HRP-Conjugated Anti-Rabbit IgG antibody and HRP-Conjugated Anti-Mouse IgG Antibody alone in different wells (without the primary antibodies). Both positive and negative controls should be performed in the same plate with the target protein target experiments.

5.

Accuracy and Precision: Each condition should be performed in duplicate or in triplicate.

1.

Seed 200 µl of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µl of 10 µg/ml
Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.

2.

Incubate the cells for overnight at 37°C, 5% CO2.

3.

Treat the cells as desired.

4.

Remove the cell culture medium and rinse with 200 µl of 1x TBS, twice.

5.

Fix the cells by incubating with 100 µl of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells. During the incubation, the plates should be sealed with Parafilm.
Note: Fixing Solution is volatile. Wear appropriate personal protection equipment (mask, gloves and glasses) when using this chemical.

6.

Remove the Fixing Solution and wash the plate 3 times with 200 µl 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
Note: For all wash steps, tap the plate gently on absorbent papers to remove the solution completely.

7.

Add 100 µl Quenching Buffer and incubate for 20 minutes at room temperature.

8.

Wash the plate 3 times with 1x Wash Buffer for 5 minutes at a time, with gentle shaking on the shaker.

9.

Add 200 µl of Blocking Buffer and incubate for 1 hour at room temperature

10.

Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes at a time, with gentle shaking on the shaker.

11.

Add 50 µl of each of the 1x Primary Antibodies to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature with gentle shaking on the shaker.

12.

Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes at a time, with gentle shaking on the shaker.

13.

Add 50 µl of 1x Secondary Antibodies (HRP-Conjugated Anti-Rabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature with gentle shaking on the shaker.
Note: Add HRP-Conjugated Anti-Rabbit IgG Antibody to the wells incubated with the Primary Antibodies and add HRP-Conjugated Anti-Mouse IgG Antibody to the wells incubated with Anti-GAPDH Antibody (mouse, monoclonal).

14.

Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes at a time, with gentle shaking on the shaker.

15.

Add 50 µl of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark with gentle shaking on the shaker.
Note: Ready-to-Use Substrate is a light-sensitive reagent. Keep away from light.

16.

Add 50 µl of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

17.

After finishing reading the absorbance at 450 nm, wash the plate twice with 200 µl of Wash Buffer and twice with 200 µl of 1x TBS for 5 minutes each. Tap the plates on paper towel to remove the excess liquid. Let plate air dry for 5 minutes at room temperature.

18.

Add 50 µl of Crystal Violet Solution to each well, incubate for 30 minutes at room temperature on the shaker.
Note: Crystal Violet is an intense stain. Avoid contact with skin and clothing.

19.

Tip off Crystal Violet solution into beaker. Wash plate by dipping into bucket of water in the sink with the water continuing to run. Carefully rinse the wells in ddH2O until no more colour comes off the wells. Allow the plate to dry for 30 minutes.

20.

Add 100 µl of SDS Solution into each well and incubate on the shaker at room temperature for 1 hour.

21.

Read absorbance at 595 nm with microplate reader. If absorbance is too high, the solubilized Crystal Violet Solution can be diluted 10 times with ddH2O on a separate 96-well plate.

GAPDH Normalization

The OD450 values obtained for the target protein can be normalized sing the OD450 values obtained for GAPDH.

Crystal Violet Staining Normalization

The measured OD450 readings can be normalized using the OD595 values via the proportion, OD450/OD595.