Phospho-specific ELISA Protocol

Introduction

“PhosphoELISA” kits are phospho-specific sandwich ELISA (enzyme-linked immunosorbent assay) kits that allow for quantitative measurement of protein inside the cell. Phospho-specific ELISA kits can be used during signaling studies to measure phosphorylation, modification, cleavage site-specific and total proteins. For more information visit our Phospho-Specific ELISA Kit page.

We offer a large menu of Thermo Scientific Pierce and Novex phospho-specific kits including STAT3, CREB, PRAS40, p38, AMPK alpha, PTK2 and Caspase 3 signaling. These kits are validated and manufactured to help ensure excellent quality and reproducibility. The kits meet quality-controlled specifications for criteria such as sensitivity, dynamic range, precision, specificity, recovery and lot-to-lot consistency. See our ELISA Selection Tool to find a kit to help with your research needs.

The ELISA method is a benchmark for quantitation of antigens. ELISAs are adaptable to high-throughput screening because results are rapid, consistent and relatively easy to analyze. The best results have been obtained with the sandwich format, utilizing highly purified, pre-matched capture and detection antibodies. The resulting signal provides data which is very sensitive and highly specific.

Phospho-specific protocols (Figure 1) begin with a capture antibody, specific for a protein of interest, coated onto the wells of microplates. Samples, including a standard containing protein of interest, control specimens, and unknowns, are pipetted into wells. During the first incubation, the protein antigen binds to the capture antibody. After washing, a detection antibody is added to the wells, this antibody binds to the immobilized protein captured during the first incubation. After removal of excess detection antibody, an enzyme-linked (i.e., HRP) secondary antibody is added and binds to the detection antibody. After a third incubation and washing to remove the excess secondary antibody, a substrate solution is added and is converted by the enzyme to a detectable form (color signal.) The intensity of this colored product is directly proportional to the concentration of antigen present in the original specimen. Thermo Scientific Pierce and Novex phospho-specific kits have been designed to enable specific detection of phosphorylation, offer great specificity and correlate well to western blot data.

Figure 1. Scheme of a typical phospho-specific protocol. Our ready-to-use Phospho-specific ELISA kits come with standard, detection antibody, HRP conjugate, chromogenic substrate and stop solution to complete all steps listed above.

Note: The protocol provided is representative of most phospho-specific ready-to-use ELISA kits. Protocols for individual kits may differ. Depending on the protein of interest, antibodies, buffers, or substrates being used, this general protocol may need to be optimized. For more information see Overview of ELISA and ELISA Development and Optimization.

Typical Phospho-specific ELISA Kit Components

• Antibody-coated 96-well microplate.
  
• Detection antibody (sometimes biotinylated).
  
• Standard.
  
• HRP conjugate (antibody or streptavidin).
  
• Diluent buffers.
  
• Wash buffer.
  
• Chromogenic substrate (i.e., TMB).
  
• Stop solution.
  
• Plate covers.

Additional Materials Required

• Cell extraction buffer (Cat. No. FNN0011).
  
• Distilled or deionized water.
  
• Absorbance-based microplate reader.
  
• Squirt wash bottle or an automated 96-well plate washer.
  
• Sample (See ELISA Sample Preparation Protocols).

Example Protocol

Run time: 4 hours — 30 minutes hands-on time.
Note: A standard curve must be run with each assay for quantification.

1. Allow all reagents to reach room temperature before use. Gently mix all liquid reagents prior to use.
   
2. Add 50–100µL of prepared standard and sample to wells. Tap gently on side of plate to thoroughly mix. Cover plate and incubate for 2 hours at room temperature.
   
   Note: Samples prepared in cell extraction buffer must be diluted 1:10 or greater in diluent buffer. The dilution chosen should be optimized for each experimental system.
   
   
3.  Thoroughly aspirate or decant solution from wells and discard the liquid.
4. Wash wells 4 times using a squirt wash bottle or an automated 96-well plate washer.
   
   Note: No matter your method of washing, (squirt bottle or automated plate washer) fill wells with at least 400µL of diluted wash buffer. Let soak for 15 to 30 seconds, and then decant the liquid. Repeat. Use this method for all subsequent wash steps.
   
   
5. Add 100µL of diluted detection antibody to wells. Tap gently on side of plate to thoroughly mix. Cover plate and incubate for 1 hour at room temperature.
6. Thoroughly aspirate or decant solution from wells and discard the liquid.
   
7. Wash wells 4 times.
   
8. Add 100µL of diluted HRP conjugate to each well. Cover plate and incubator at room temperature for 30 minutes.
   
9. Thoroughly aspirate or decant solution from wells and discard the liquid.
   
10. Wash wells 4 times.
11. Add 100µL of chromogenic substrate to each well.
    
12. Develop plate at room temperature in the dark for 30 minutes.
    
13. Add 100µL of stop solution to each well. Tap side of plate gently to mix. The solution in the wells should change from blue to yellow.
    
14. The plate must be evaluated within 30 minutes of stopping the reaction. Read the absorbance of each well at 450 and 550nm. Subtract 550nm values from 450nm values to correct for optical imperfections in the microplate.
    
15. Calculate results manually using a curve-fitting statistical software package, plot a four-parameter logistic curve fit.

Note: If using an Antibody Pair Kit, to build your own ELISA, you will need to coat the capture antibody on to a 96-well microplate yourself. For this process you will need: a 96-well microplate, capture antibody and blocking buffer (usually BSA or milk diluted in PBS). You will follow these steps before starting the above protocol:

1. Allow all reagents to reach room temperature before use. Gently mix all liquid reagents prior to use.
   
2. Add 100µL of diluted capture antibody to each well. Cover plate and incubate at 4oC overnight.
   
3. Bring plate to room temperature. Thoroughly aspirate or decant solution from wells and discard the liquid.
   
4. Wash wells 4 times.
   
5. Add 200µL of blocking buffer to each well. Cover plate and incubate at room temperature for 1 hour.
   
6. Thoroughly aspirate or decant solution from wells and discard.
   --Continue with above protocol --
   

Featured Data

Thermo Scientific Pierce and Novex phospho-specific ELISA kits are highly tested and validated. Below is data from our most popular kits. For more information about our range of phospho-specific ELISA kits please visit our Phospho-Specific ELISA Kit page.

Figure 2. ERK 1/2 [pT185/pY187] Phospho-ELISA Kit, Human. Using the above protocol samples were serially diluted and run using the ERK 1/2 [pT185/pY187] Phospho-ELISA Kit, Human (Cat. No. KHO0091). Parallelism between ERK 1/2 standard and PMA-treated Jurkat (immortalized human T lymphocyte) cell lysate was demonstrated.		Figure 3. PRAS40 [pT246] Phospho-ELISA Kit, Human. Using the above protocol samples were serially diluted and run using the PRAS40 [pT246] Phospho-ELISA Kit (Cat. No. KHO0421). Parallelism between PRAS40 standard and Jurkat (immortalized human T lymphocyte) cell lysate was demonstrated.