Prestige Antigens™ and the corresponding Prestige Antibodies are recommended to be used according to the standard blocking protocol described below.
Re-titrate the antibody to define the optimal dilution to be used in the blocking study. Optimal titration is the highest dilution possible at which antibody still shows expected staining.
Calculate 10X and 100X molar excess of the Prestige Antigen based on concentrations and MWs of antibody (150,000 Da) and Prestige Antigen (15,000 Da).
Mix calculated amounts of respective antibody and Prestige Antigen in 2 mL tube and adjust total volume to 30-50 μL with PBS.
Incubate tube containing antibody/Prestige Antigen for 30 min at room temperature, or 4 °C overnight with gentle shaking.
As a control, use the primary antibody without Prestige Antigen, or a different Prestige Antigen (not corresponding to the target protein).
Centrifuge the tube with antibody/Prestige Antigen for 15 min at maximum speed (15,000 rpm) to pellet any immune complexes.
Carefully pipette the supernatant into a new tube to prepare a working solution for immunostaining. Since the antibody is now diluted 4-12 times, the volume of antibody diluent must be adjusted accordingly.
Proceed with your experiment according to the general IHC or ICC protocol.
Pre-incubate primary antibody diluted 1:250 with corresponding Prestige Antigen (100X molar excess) in 3,5 mL block buffer for 30 min at room temperature. 100X molar excess of Prestige Antigen is calculated based on concentrations and MWs of antibody (150,000 Da) and PrEST Antigen (15,000 Da).
As a Western blot control, include a primary antibody reaction without Prestige Antigen or use a different Prestige Antigen not corresponding to your antibody target.
Add incubated antibody-Prestige Antigen mixture to the blocked membrane and incubate on a roller mixer or rocking shaker for 1 hour at room temperature.
Proceed with the Western blot according to the general Western blotting protocol.
Revised Oct 2017