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Colorimetric 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 Colorimetric 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 the HRP-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the HRP enzyme conjugated to the 2° antibody can catalyze a colorimetric reaction upon substrate addition.

Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are described: 1) a monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values. 2) Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method is used to determine cell density.

After staining, the results can be analyzed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. 3) If a phosphorylated target is being detected, an antibody against the non- phosphorylated counterpart will be provided for normalization purposes. The absorbance values obtained for the non-phosphorylated target can be used to normalize the absorbance values for the phosphorylated target.

Materials (Cell-Based Phospho):

  • Reagent
  • 96-Well 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
  • HRP-Conjugated Anti-Rabbit IgG Antibody
  • HRP-Conjugated Anti-Mouse IgG Antibody
  • Primary Antibody Diluent
  • Ready-to-use Substrate
  • Stop Solution
  • Crystal Violet Solution
  • SDS Solution
  • Adhesive Plate Sealers


  • Quantity
  • 2 Plates
  • 24 ml(1x)
  • 24 ml (10x)
  • 50 ml (1x)
  • 50 ml (15x)
  • 60 μl (100x)
  • 60 μl (100x)
  • 60 μl (100x)
  • 12 ml (1x)
  • 12 ml (1x)
  • 12 ml (1x)
  • 12 ml (1x)
  • 12 ml (1x)
  • 12 ml (1x)
  • 24 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 1x Primary Antibodies or Anti-GAPDH Antibody) 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.

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 Primary Antibodies (rabbit, polyclonal) 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.

Optional

Crystal Violet binds to cell nuclei and gives absorbance eadings proportional to cell counts at 595 nm.

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 mins 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 color 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.