Quantitative Dot Blot to Estimate Target Proteins in a Tissue Lysate
Transcript
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.
