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Fluorometric Cell-Based ELISA - Protocol

The Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor protein expression profile in cells. The kit can be used for measuring the relative amounts of in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on protein expression.

Introduction:

The Fluorometric Cell-Based ELISA Kit allows for the detection of various target proteins and the effects that certain stimulation conditions have on target protein expression in different cell lines. Qualitative determination of target protein concentration is achieved by an indirect ELISA format.

In essence, the target protein is captured by target-specific primary (1°) antibodies while Dye 1-conjugated and Dye 2-conjugated secondary (2°) antibodies bind the Fc region of then 1° antibody. Through this binding, the dye conjugated to the 2° antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method.

A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target RFU values. If a phosphorylated target is being detected, an antibody against the non-phosphorylated counterpart will be provided for normalization purposes. The RFU values obtained for the non-phosphorylated target can be used to normalize the RFU value for the phosphorylated target.

Materials (Cell-Based Phospho):

  • Reagent
  • 96-Well Black Cell Culture Clear-Botom Microplate
  • 10x TBS
  • Quenching Buffer
  • Blocking Buffer
  • 15x Wash Buffer
  • 100x Anti-Phosphorylated Target Antibody
  • 100x Anti-Non Phosphorylated Target Antibody
  • 100x Anti-GAPDH
  • Dye1-Conjugated Anti-Rabbit IgG Antibody
  • Dye2-Conjugated Anti-Mouse IgG Antibody
  • Primary Antibody Diluent
  • Adhesive Plate Sealers


  • Quantity
  • 2 Plates
  • 24 ml (1x)
  • 24 ml (10x)
  • 50 ml (1x)
  • 50 ml (15x)
  • 60 μl (100x)
  • 60 μl (100x)
  • 110 μl (100x)
  • 6 ml (1x)
  • 6 ml (1x)
  • 12 ml (1x)
  • 2 Sheets

Assay Protocol:

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 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 gente 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 “Primary Antibody Mixture P” to corresponding wells for Phosphorylated target detection. Add 50 ul of “Primary Antibody Mixture NP” to the corresponding wells for total BCAR1 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 5 minutes at a 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.