Griffin:Antibody Peptide Neutralization

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Contents

Overview

Blocking (neutralizing) peptides are available as negative controls for all Santa Cruz Biotechnology, Inc. affinity-purified rabbit and goat polyclonal antibodies and monoclonal antibodies raised against peptide antigens. Antibody binding to antigen may be blocked/competed by pre-absorption with the blocking peptide.

This is a negative control experiment since the anticipated staining or band pattern, if it is specific due to the antigen binding capacity of the primary antibody, should disappear upon peptide blocking.

The blocking peptide (peptide antigen) is a negative control for an antibody. Antibody binding to the original antigen will neutralize the IgG. Typically peptide neutralization is performed for western blotting, IF, and IHC applications.

Peptide Solution

100 ug/0.5 ml in 1X PBS, 100 ug BSA (0.2%), 0.1% Sodium Azide

Protocol

1. Titrate antibody to the dilution that gives the desired positive result.

2. Determine the weight of antibody used.

3. Pipet the antibody & blocking peptide at a 1:5 weight to weight ratio in a microfuge tube (5 fold excess peptide to IgG). Incubate 2 hours at room temperature or overnight at 4ºC.

  • For example: If you use 1 ug of antibody in your experiment, then combine with 5 ug of peptide.

4. Dilute antibody/peptide mixture into appropriate blocking buffer. Proceed with experiment.

References

Santa Cruz Biotechnology Inc.

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