Pre-adsorbed secondary antibodies
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Minimize non-specific binding and high background staining.
Reviewed July 6, 2021
Pre-adsorption (also referred to as cross-adsorption) is an extra purification step introduced to increase the specificity of a secondary antibody. The solution containing secondary antibodies is passed through a column matrix containing immobilized serum proteins from potentially cross reactive species. Non-specific secondary antibodies are retained in the column, while highly specific secondaries flow through.
Figure 1: In this example, a solution of secondary antibodies recognizing rabbit IgG light chains is passed through a matrix containing immobilized serum proteins from potentially cross-reactive species such as sheep and bovine IgGs. Only antibodies highly specific to rabbit IgG light chain will flow through the column, while secondary antibodies cross-reacting with sheep or bovine IgG light chains will remain bound to the proteins in the column.
Advantages of pre-adsorbed secondary antibodies
We always recommend using pre-adsorbed (cross-absorbed) secondary antibodies to minimize non-specific background when working with tissues and cells. This means pre-adsorbed antibodies are useful in many different experiments, however they are most useful for:
- Multicolor experiments: When several primary and their corresponding secondary antibodies are used simultaneously, the secondary antibody should be pre-adsorbed against the species in which the other primary antibodies have been raised to reduce the risk of cross reactivity.
Example: Use an anti-rabbit Alexa Fluor® 488 pre-adsorbed secondary against mouse serum and an anti-mouse Alexa Fluor® 647 pre-adsorbed secondary against rabbit serum when performing a double staining experiment using primary antibodies raised in rabbit and mouse respectively.
- IHC experiments on samples with abundant amounts of endogenous immunoglobulins (Igs): The secondary antibody should be pre-adsorbed against the same species the sample originated from to reduce the risk of cross reactivity between the secondary antibody and endogenous Igs.
Example: Use an anti-mouse HRP pre-adsorbed secondary antibody against human serum when staining human tissues or cell lines.
Pre-adsorbed Alexa Fluor® conjugated secondary antibodies
All pre-adsorbed Alexa Fluor® secondary antibodies listed below are suitable for use in ICC/IF, ELISA, IHC-P, IHC-Fr and flow cytometry.
Pre-adsorbed HRP conjugated secondary antibodies
Please note some of our pre-adsorbed HRP secondary antibodies are only suitable for use in specific applications, these are listed below.
| Product Name | Applications |
| Anti-mouse HRP (IgG H&L) (ab97040) | IHC-P, ELISA, WB |
| Anti-rabbit HRP (IgG H&L) (ab97080) | ICC, IHC-P, ELISA, WB |
| Anti-rat HRP (IgG H&L) (ab7097) | ICC/IF, IHC-P, IHC-Fr, WB, ELISA, Dot Blot |
| Anti-goat HRP (IgG H&L) (ab97120) | ICC, IHC-P, ELISA, WB |
| Anti-chicken HRP (IgY H&L) (ab7118) | WB, Immunomicroscopy, ICC/IF, Dot Blot, ELISA, IHC-P, IHC-Fr |
Pre-adsorbed biotinylated secondary antibodies
Please note some of our pre-adsorbed biotinylated secondary antibodies are only suitable for use in specific applications, these are listed below
| Product Name | Applications |
| Anti-mouse Biotin (IgG H&L) (ab7074) | ICC/IF, Dot Blot, ELISA, IHC-P, IHC-Fr, Immunomicroscopy, WB |
| Anti-rabbit Biotin (IgG H&L) (ab7082) | ICC/IF, WB, ELISA, Immunomicroscopy, Dot Blot, IHC-P, IHC-Fr |
| Anti-rat Biotin (IgG H&L) (ab7096) | ICC/IF, WB, ELISA, Immunomicroscopy, Dot Blot, IHC-P, IHC-Fr |
| Anti-goat Biotin (IgG H&L) (ab7124) | ICC/IF, Dot Blot, ELISA, IHC-P, IHC-Fr, Immunomicroscopy, WB |
| Anti-chicken Biotin (IgY H&L) (ab7117) | ICC/IF, Dot Blot, ELISA, IHC-P, IHC-Fr, Immunomicroscopy, WB |
Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.