Fluorometric Cell-Based ELISA Kit Protocol

10x TBS

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

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.

15x 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.

Primary Antibody Diluent

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

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.

100x Anti-Phospho-Target Primary Antibody

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-Target Primary Antibody

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.

Dye-1 Anti-Rabbit IgG Secondary Antibody

Dye-1 Conjugated Anti-Rabbit IgG antibody is the secondary antibody to detect the target bound, primary rabbit antibodies. The solution is light sensitive. Please store and handle in the dark.

Dye-2 Conjugated -Mouse IgG Secondary Antibody

Dye-2 Conjugated Anti-Mouse IgG Antibody is used as the secondary antibody to detect the target bound, primary mouse antibodies. The solution is light sensitive. Please store and handle in the dark.

Primary Antibody Mixture P

Immediately before use, add 50 ul of the 100x Anti-Phosphorylated Protein Antibody and 50 ul of 100x Anti-GAPDH Antibody to 4,900 ul of Primary Antibody Diluent (for one plate, 96 wells). Gently mix and label the tube as “Primary Antibody Mixture P”.

Primary Antibody Mixture NP

Immediately before use, add 50 ul of 100x Anti-Target Protein Antibody and 50 ul of 100x Anti-GAPDH Antibody to 4,900 ul of Primary Antibody Diluent (for one plate, 96 wells). Gently mix and label the tube as “Primary Antibody Mixture NP”.

Secondary Antibody Mixture

Immediately before use, for one plate, mix 3 ml of Dye-1 Conjugated Anti-Rabbit IgG Antibody and 3 ml of Dye-2 Conjugated Anti-Mouse IgG Antibody. Gently mix and label tube as “Secondary Antibody Mixture”. This solution is light sensitive. Please store and handle in the dark.

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 adjustments are required.
a. 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 prior to cell seeding.
b. 8% Formaldehyde (not included) instead of 4% is required.

2.

Cell Number and Sensitivity: The number of cells plated onto the 96-well plates depends on the expression level of the target protein in the cells, cell size, treatment conditions and incubation time. The cells used for testing should be around 75-90% confluency.

3.

Cell Treatment: The cells can be treated with inhibitors, activators, stimulators (ie. chemicals, proteins/peptides) or a combination of the substances listed above. Stimulation of cells should be controlled. Non-stimulated wells of cells should be included within the same plate.
Note: Plate cover must be off during treatment of cells.
Note: Cell death via various stimulations may occur and should be factored into experimental design.

4.

Positive, Negative and Blank Controls:
a. Positive Control: Mouse Anti-GAPDH Antibody is an internal positive control used to normalize the RFU values of the target protein in each well.

b. Negative Control: Incubation of only Secondary Antibody Mixture. 50 ul of Primary Antibody Diluent is incubated instead of Primary Antibody Mixture in these wells.

c. Blank Control: Incubation of 50 ul Primary Antibody Diluent instead of any Antibody Mixture.

The Positive, Negative and Blank controls should be performed in the same plate with the Target Protein experiments.

5.

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

1.

Seed 200 µl of desired cell concentration in culture medium into each well of the 96-well plates. 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.
Note: Treatment of cells may result in cell death should be considered prior to treatment.
Note: Vigorous pipetting may knock cells off of the plate. Utilize the well walls to dispense/aspirate gently at each step.

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.
a.4% formaldehyde is used for adherent cells.
b.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 3 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 3 minutes each time with gentle shaking on the shaker.

9.

Dispense 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 3 minutes at a time with gentle shaking on the shaker.

11.

Add 50 µl of “Primary Antibody Mixture P” to corresponding wells for target protein detection. Add 50 ul of “Primary Antibody Mixture NP” to the corresponding wells for total protein detection. Cover the plate 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.

12.

Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time with gentle shaking on the shaker.

13.

Add 50 ul of “Secondary Antibody Mixture” to corresponding wells and incubate for 1.5 hours at room temperature with gentle shaking.
Note: The plates should be kept in the dark for each step after addition of “Secondary Antibody Mixture”.

14.

Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes at a time, with gentle shaking on the shaker. Afterwards, rinse once with 200 ul of 1x TBS. Keep the plate(s) in the dark during wash.

15.

Read the plate(s) at Ex/Em: 651/667 (Dye 1) and 495/521 (Dye 2). Shield plates from direct light exposure.

Anti-Target Protein Primary Antibody Normalization

The RFU values obtained for the phosphorylated target protein can be normalized using the RFU values obtained for the non-phosphorylated target protein via the proportion, RFU (Anti-phospho target antibody)/RFU (Anti-target antibody). This assumes that both RFUs included in the equation are derived from the same read under the same excitation and emission wavelengths.

GAPDH Normalization

The RFU values obtained for the target protein (phosphorylated and nonphosphorylated) can be normalized using the RFU values obtained for GAPDH. Anti-GAPDH Stimulated RFU/ Anti-GAPDH Non-Stimulated may be used as a ratio in determining Cell Density. This ratio may be used as a multiplier for Anti-Target Phospho Stimulated RFU values in comparison to Anti-Target Non-Stimulated RFU values. The calculated ratios of RFU values are meant to be a reference point for qualitative measurement of levels of expression between wells.