Cell-Based ELISA Assay

ELISA is a more quantitative method than western blotting and shows great utility in the study of modulating kinase activity and function. This microplate-based assay typically utilizes a capture antibody specific for the desired protein, which is independent of the phosphorylation status. The target protein, either purified or a component of a complex heterogeneous sample such as cell lysate, is then bound to the antibody-coated plate. A detection antibody directly against the phosphorylation site to be analyzed is then added. This assay is typically designed using colorimetric or fluorescent detection. The intensity of the resulting signal is directly proportional to the concentration of phosphorylated protein present in the original sample.

Cell-based ELISA is a convenient, lysate-free, high throughput and sensitive assay that can measure the relative amount of protein phosphorylation in cells. The method can also be used for monitoring the effects of various treatments, activators or inhibitors have on phosphorylation.

Figure 1. The workflow of cell-based ELISA.

Materials and Equipment

Table 1. List of ingredients.

Assay Procedure

• Seed 100 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, coating the plates with 100 μL of 10 μg/mL poly-l-lysine to each well and then follow the manufacturer’s instructions.
  Note: The optimal cell number plated onto the 96-well plates depends on the expression level of Met protein in the cells, cell size, treatment conditions and incubation time.
• Incubate the cells for overnight at 37°C, 5% CO₂.
• Treat the cells as desired.
  Note: The cells can be treated with inhibitors, activators, stimulators or a combination of the substances listed above. The cells can also be treated with UV and serum starvation to meet the needs of the end-user.
• Remove the cell culture medium and rinse with 200 μL of 1X TBS, twice.
• 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.
• Remove the fixing solution and wash the plate 3 times with 200 μL 1X wash buffer for 5 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.
• Add 100 μL quenching buffer and incubate for 20 minutes at room temperature.
  Note: The quenching buffer is used to inactivate the endogenous peroxidase activity and minimize the background response.
• Wash the plate 3 times with 1X wash buffer for 5 minutes at a time, with gentle shaking on the shaker.
• Add 200 μL of blocking buffer and incubate for 1 hour at room temperature.
  Note: Blocking buffer is used to block additional binding sites in each well.
• 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 primary antibodies (anti-Met antibody and/or anti-GAPDH antibody) to the corresponding wells, cover with parafilm and incubate for 16 hours (overnight) at 4°C.
  Note: If the target expression is known to be high, incubate for 2 hours at room temperature with gentle shaking on the shaker.
• 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 secondary antibodies (HRP-conjugated anti-rabbit IgG and/or HRP-conjugated anti-mouse IgG) 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 to the wells incubated with anti-Met and add HRP-conjugated anti-mouse IgG to the wells incubated with anti-GAPDH antibody.
• 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 substrate to each well and incubate for 30 minutes at room temperature in the dark with gentle shaking on the shaker.
  Note: 1) Keep away from light as the substrate is light-sensitive. 2) Substrate must be warmed to room temperature before use.
• Add 50 μL of stop solution to each well and read OD at 450 nm immediately using the microplate reader.

For research use only. Not for any other purpose.