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Prestige Antigens™-Blocking Protocol

Prestige Antigens and the corresponding Prestige Polyclonals or Monoclonals are recommended to use according to the standard Prestige Antigen-blocking protocol described below.

Prestige Antigens in Pre-Adsorption for IHC or ICC Experiments

Re-titrate the antibody in order 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 2ml 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 max 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 IHC experiment according to the general IHC, or ICC, protocol.

Prestige Antigens in Pre-Adsorption for WB Experiments

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 Prestige Antigen (15,000 Da).

As 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 WB experiment according to the general WB protocol.

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