pERK antibody validation standard

Biotinylated Antibody

Calculate the total volume needed for the assay by multiplying 0.1 mL/well and the number of wells required. Add 2-3 extra wells to the calculated number of wells to account for possible pipetting errors. Generate the required volume of diluted antibody by performing a 1:100 dilution (For each 1 μL concentrated antibody, add 99 μL antibody dilution buffer) and mixing thoroughly.

Avidin-Biotin-Peroxidase (ABC)

Diluted ABC solution has to be prepared freshly. Calculate the total volume needed for the assay by multiplying 0.1 mL/well and the number of wells required. Add 2-3 extra wells to the calculated number of wells to account for possible pipetting errors. Generate the required volume of diluted ABC solution by performing a 1:100 dilution (For each 1 μL concentrated ABC solution, add 99 μL ABC dilution buffer) and mixing thoroughly.

Direct ELISA protocol

This is a general protocol in which antigen coating and blocking may not be required if the wells have been pre-adsorbed with the antigen by the manufacturer.

Antigen Coating

• Dilute purified antigens to a final concentration of 1-10 μg/ml in bicarbonate/carbonate antigen-coating buffer (100 mM NaHCO3 in deionized water; pH adjusted to 9.6). Pipette 100 μL of diluted antigen to each well of a microtiter plate.
• Cover the plate with adhesive plastic and incubate at 4°C overnight or 37°C for 30 minutes.
• Remove the coating solution and wash the plate 3 times with 200 μL PBS buffer with for 5 minutes each time. The coating/washing solutions can be removed by flicking the plate over a sink. The remaining drops can be removed by patting the plate on a paper towel or by aspiration. Do not allow the wells to dry out at any time.

Blocking

• Pipette 200 μL blocking buffer (5% w/v non-fat dry milk in PBS buffer) per well to block residual protein-binding sites. Alternatively, BSA or BlockACE can be used to replace non-fat dry milk.
• Cover the plate with adhesive plastic and incubate for 1-2 hour(s) at 37°C (or at 4°C overnight).
• Remove the blocking solution and wash the plate twice with 200 μL PBS for 5 min each time. Flick the plate and pat the plate as described in the coating step.

Reagent Preparation

Prepare for the diluted standard solutions as shown on above.

Primary Antibody Incubation

• Serially dilute the conjugated primary antibody with blocking buffer immediately before use. The optimal dilution should be determined by a titration assay according to the antibody manufacturer.
• Pipette 100 μL of diluted secondary antibody solution to each well.
• Cover the plate with adhesive plastic and incubate for 2 hours.
• Remove the content in the wells and wash them 3 times with 200 μL PBS buffer for 5 min each time. Flick and pat the plate as described in the coating step.

Substrate Preparation

Prepare the substrate solution immediately before use or bring the pre-made substrate to room temperature. The two widely used enzymes for signal detection are horse radish peroxidase (HRP) and alkaline phosphatase (AP), and their corresponding substrates, stopping solutions, detection absorbance wavelengths and colour developed are as follows:

TMB: 3,3’,5,5’-tetramethylbenzidine; pNPP: p-nitrophenyl-phosphate

Note:

• The TMB substrate must be kept at 37°C for 30 min before use.
• Hydrogen peroxide can also act as a substrate for HRP.
• Sodium azide is an inhibitor of HRP. Do not include the azide in buffers or wash solutions if HRP-labeled conjugate is used for detection.

Signal Detection

• Pipette 90 μL of substrate solution to the wells with the control and standard solutions.
• Incubate the plate at 37°C in the dark. If TMB is used, shades of blue will be observed in the wells with the most concentrated solutions. Other wells may show no obvious colour.
• Colour should be developed in positive wells after 15 min. After sufficient colour development, pipette 100 μL of stopping solution to the wells (if necessary).
• Read the absorbance (OD: Optical Density) of each well with a plate reader.

Data Analysis

• Prepare a standard curve using the data produced from the diluted standard solutions. Use absorbance on the Y-axis (linear) and concentration on the X-axis (log scale).
• Interpret the sample concentration from the standard curve.

Indirect ELISA protocol

This is a general protocol in which antigen coating and blocking may not be required if the wells from the manufacturer have been pre-adsorbed with the antigen.

Antigen Coating

• Dilute the purified antigens to a final concentration of 1-10 μg/mL in bicarbonate/carbonate antigen-coating buffer (100 mM NaHCO3 in deionized water; pH adjusted to 9.6).
• Pipette 100 μL of diluted antigen to each well of the microtiter plate.

Blocking

• Pipette 200 μL blocking buffer (5% w/v non-fat dry milk in PBS buffer) per well to block residual protein-binding sites. Alternatively, BSA or BlockACE can be used to replace the non-fat dry milk.
• Cover the plate with adhesive plastic and incubate for 1-2 hour(s) at 37°C or at 4°C overnight.
• Remove the blocking solution and wash the plate twice with 200 μL PBS for 5 minutes each time. Flick and pat the plate as described in the coating step.

Reagent Preparation

• Prepare for the diluted standard solutions as shown on above.

Primary Antibody Incubation

• Serially dilute the primary antibody of choice with blocking buffer. The optimal dilution should be determined by a titration assay according to the antibody manufacturer.
• Pipette 100 μL of each diluted antibody per well. Repeat in duplicate or triplicate for accuracy. The negative control should be species- and isotype-matched, as well as non-specific immunoglobulin diluted in PBS buffer.
• Cover the plate with adhesive plastic and incubate for 1 hour at 37°C or 2 hours at room temperature. These incubation times should be sufficient to achieve a strong signal. However, if a weak signal is observed, perform incubation overnight at 4°C to obtain a stronger signal.
• Remove the diluted antibody solution and wash the wells 3 times with 200 μL PBS for 5 minutes each time. Flick the plate and pat the plate as described in the coating step.

Secondary Antibody Incubation

• Serially dilute the conjugated secondary antibody with blocking buffer immediately before use. The optimal dilution should be determined by a titration assay according to the antibody manufacturer.
• Pipette 100 μL of diluted secondary antibody solution to each well.
• Cover the plate with adhesive plastic and incubate for 2 hours at room temperature.
• Remove the content in the wells and wash them 3 times with 200 μL PBS buffer for 5 minutes each time. Flick and pat the plate as described in the coating step.

Substrate Preparation

Prepare the substrate solution immediately before use or bring the pre-made substrate to room temperature. The two widely used enzymes for signal detection are horse radish peroxidase (HRP) and alkaline phosphatase (AP), and their corresponding substrates, stopping solutions, detection absorbance wavelengths and colour developed are as follows:

TMB: 3,3’,5,5’-tetramethylbenzidine; pNPP: p-nitrophenyl-phosphate

Note:

• The TMB substrate must be kept at 37°C for 30 min before use.
• Hydrogen peroxide can also act as a substrate for HRP.
• Sodium azide is an inhibitor of HRP. Do not include the azide in buffers or wash solutions if HRP-labeled conjugate is used for detection.

Sandwich ELISA protocol

Capture Antibody Coating

• Dilute the capture antibody to a final concentration of 1-10 μg/mL in bicarbonate/carbonate antigen-coating buffer (100 mM NaHCO3 in deionized water; pH adjusted to 9.6).
• Pipette 100 μL of diluted antibody to each well of a microtiter plate.
• Cover the plate with adhesive plastic and incubate at 4°C overnight (or 37°C for 30 min).
• Remove the coating solution and wash the plate 3 times with 200 μL PBS with for 5 minutes each time. The coating/washing solutions can be removed by flicking the plate over a sink. The remaining drops can be removed by patting the plate on a paper towel or by aspiration. Do not allow the wells to dry out at any time.

Blocking

• Pipette 200 μL blocking buffer (5% w/v non-fat dry milk in PBS buffer; Alternatively, BSA or BlockACE can be used to replace non-fat dry milk.) per well to block residual protein-binding sites.
• Cover the plate with adhesive plastic and incubate for 1-2 hour(s) at 37°C (or at 4°C overnight).
• Remove the blocking solution and wash the plate twice with 200 μL PBS for 5 minutes each time. Flick and tap the plate as described in the coating step.

Reagent Preparation

Prepare biotinylated antibody and ABC solutions as described previously for the diluted standard solutions.

Sample (Antigen) Incubation

• Serially dilute the sample with blocking buffer immediately before use. The optimal dilution should be determined by a titration assay according to the antibody manufacturer.
• Pipette 100 μL of each of the diluted sample solutions and control to each empty well. Repeat in duplicate or triplicate for accuracy. The negative control should be species- and isotype-matched, as well as non-specific immunoglobulin diluted in PBS buffer.
• Cover the plate with adhesive plastic and incubate for 2 hours at room temperature.
• Remove the content in the wells and wash them 3 times with 200 μL PBS buffer for 5 minutes each. Flick the plate and pat the plate as described in the coating step.

Biotinylated Antibody Incubation

• Pipette 100 μL of diluted antibody to the control, standard solutions and diluted samples.
• Cover the plate with adhesive plastic and incubate for 1 hour at 37°C (or 2 hours at room temperature). These incubation times should be sufficient to receive a strong signal. However, if a weak signal is observed, perform incubation overnight at 4°C for a stronger signal.
• Remove the content in the wells and wash them 3 times with 200 μL PBS for 5 min each time. Flick the plate and pat the plate as described in the coating step.

ABC Incubation

• Pipette 100 μL of diluted ABC solution to the wells with control, standard solutions and diluted samples.
• Cover the plate with adhesive plastic and incubate for 30 minutes at 37°C.
• Remove the content in the wells and wash them 3 times with 200 μL PBS buffer for 5 minutes each. Flick the plate and pat the plate as described in the coating step.

Substrate Preparation

Prepare the substrate solution immediately before use or bring the pre-made substrate to room temperature. The two widely used enzymes for signal detection are horse radish peroxidase (HRP) and alkaline phosphatase (AP), and their corresponding substrates, stopping solutions, detection absorbance wavelengths and colour developed are as follows:

TMB: 3,3’,5,5’-tetramethylbenzidine; pNPP: p-nitrophenyl-phosphate

Note:

• The TMB substrate must be kept at 37°C for 30 minutes before use.
• Hydrogen peroxide can also act as a substrate for HRP.
• Sodium azide is an inhibitor of HRP. Do not include the azide in buffers or wash solutions if HRP-labeled conjugate is used for detection.

Signal Detection

• Pipette 90 μL of substrate solution to the wells with the control, standard solutions and diluted samples.
• Incubate the plate at 37°C in the dark. If TMB is used, shades of blue will be observed in the wells with the most concentrated solutions. Other wells may show no obvious colour.
• A colourimetric signal should be developed in positive wells after 15 minutes. Stop the reaction by adding 100 μL of stop solution.
• Read the absorbance (OD: Optical Density) of each well with a plate reader.

Data Analysis

• Prepare a standard curve using the data produced from the diluted standard solutions. Use absorbance on the Y-axis (linear) and concentration on the X-axis (log scale). Competitive ELISA yields an inverse curve: Higher values of antigen in the samples yield a smaller amount of colour change.
• Interpret the sample concentration from the standard curve.

Competitive ELISA protocol

This is a general protocol in which antigen coating and blocking may not be required if the wells have been pre-adsorbed with the antigen by the manufacturer.

Antigen Coating

• Dilute purified antigens to a final concentration of 20 μg/ml in bicarbonate/carbonate antigen-coating buffer (100 mM NaHCO3 in deionized water; pH adjusted to 9.6).
• Pipette 100 μL of diluted antigen to each well of a microtiter plate.
• Cover the plate with adhesive plastic and incubate at 4°C overnight or 37°C for 30 minutes.
• Remove the coating solution and wash the plate 3X with 200 μL PBS buffer with for 5 minutes each time. The coating/washing solutions can be removed by flicking the plate over a sink. The remaining drops can be removed by patting the plate on a paper towel or by aspiration. Do not allow the wells to dry out at any time.

Blocking

• Pipette 200 μL blocking buffer (5% w/v non-fat dry milk in PBS buffer) per well to block residual protein-binding sites. Alternatively, BSA or BlockACE can be used to replace non-fat dry milk.
• Cover the plate with adhesive plastic and incubate for 1-2 hour(s) at 37°C or at 4°C overnight.
• Remove the blocking solution and wash the plate twice with 200 μL PBS for 5 minutes each time. Flick and pat the plate as described in the coating step.

Reagent Preparation

Prepare for the diluted standard solutions as shown on p.9.

Sample Incubation

• Serially dilute the sample with blocking buffer immediately before use. The optimal dilution should be determined by a titration assay according to the antibody manufacturer.
• Pipette 100 μL of diluted sample to each well.
• Cover the plate with adhesive plastic and incubate for 2 hours at room temperature.
• Remove the content in the wells and wash them three times with 200 μL PBS buffer for