Quantitative Dot Blot to Estimate Target Proteins in a Tissue Lysate

Quantitative dot blot — or QDB — is a useful technique to quantify the concentration of target proteins in a sample. Once the sample is immobilized on a polymeric membrane, the proteins can be detected via immunoblotting.

To begin, take a QDB plate — a multi-well plate with a nitrocellulose membrane attached to the bottom of each well. Add the sample — a tissue lysate containing the target protein — at the center of the membrane inside the well.

Allow the sample to dry. The target protein binds to the membrane via non-covalent interactions.

Add a blocking solution. The proteins in the solution attach to the unbound sites on the membrane, preventing the non-specific binding of detection reagents.

Next, add a solution containing primary antibodies that bind to the target protein in the sample. Wash to remove unbound antibodies.

Then, add a secondary antibody solution. These molecules — labeled with a peroxidase enzyme — bind to the protein-bound primary antibodies. Wash to remove unbound antibodies.

Finally, add a solution containing a chemiluminescent substrate and hydrogen peroxide. Utilizing hydrogen peroxide, the antibody-bound peroxidase oxidizes the substrate and emits light. Using a plate reader, measure the luminescence intensity of the sample.

Generate a standard luminescence curve using a serial dilution with known concentrations of the target protein. Plot the measured luminescence value of the sample on the curve to determine the target protein concentration.

In this procedure, place an empty pipette tip box under the QDB plate to avoid the bottom of the plate touching the table surface. Load up to 2 microliters of the sample to the membrane center at the bottom of an individual unit of the QDB plate.

Never allow the bottom of the QDB plate touching any surface, while loading, and do not load too much for individual well. You are experience no more than 4 microliter per well.

Next, leave the loaded QDB plate for one hour at room temperature, or leave the loaded plate at 37 degrees Celsius for fifteen minutes in a well-ventilated space. To block the plate, dip the QDB plate in the transfer buffer and gently shake it for 10 seconds.

After that, rinse the QDB plate gently with TBST three times and then wash the plate for five minutes in TBST under constant shaking. Subsequently, block the QDB plate with blocking buffer for one hour under constant shaking. Now, dilute the primary antibody in the blocking buffer at a chosen concentration, and add 100 microliters of it to each individual well of an ordinary 96-well plate.

Insert the QDB plate into the 96-well plate, and incubate the combined plates either for two hours at room temperature, or overnight at 4 degrees Celsius under constant shaking. After that, rinse the plate gently with TBST three times, before it is washed with TBST three times -- each time for five minutes under constant shaking.

Next, dilute the secondary antibody in the blocking buffer at the chosen concentration and aliquot 100 microliters into each well of a 96-well plate. Then, incubate the QDB plate inside the loaded 96-well plate for one hour at room temperature under constant shaking. Rinse the QDB plate gently three times with TBST, and then, wash the plate three times, five minutes each with TBST under constant shaking.

For quantification, prepare ECL substrate and aliquot into a 96-well plate at 100 microliters per well. Then, insert the QDB plate inside the 96-well plate for two minutes under constant shaking. After that remove the QDB plate from the 96-well plate and shake briefly to remove the excess liquid. Subsequently, place the plate onto a white microtiter plate. Next, turn on the microplate reader and select plate with cover on the user interface, before placing the combined plates inside the microplate reader for quantification.