Griffin:Antibody Peptide Neutralization: Difference between revisions
No edit summary |
|||
| (5 intermediate revisions by the same user not shown) | |||
| Line 2: | Line 2: | ||
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. | 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 | 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 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=== | ===Peptide Solution=== | ||
100 ug/0.5 ml in 1X PBS, 100 ug BSA, 0.1% Sodium Azide | 100 ug/0.5 ml in 1X PBS, 100 ug BSA (0.2%), 0.1% Sodium Azide | ||
==Protocol== | ==Protocol== | ||
| Line 16: | Line 16: | ||
2. Determine the weight of antibody used. | 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 | 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 | 4. Dilute antibody/peptide mixture into appropriate blocking buffer. Proceed with experiment. | ||
==References== | ==References== | ||
[http://www.scbt.com/protocol_peptide_neutralization.html Santa Cruz Biotechnology Inc.] | [http://www.scbt.com/protocol_peptide_neutralization.html Santa Cruz Biotechnology Inc.] | ||
Revision as of 15:11, 24 February 2010
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.
