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Integrin

Pre-adsorbed secondary antibodies

Related

  • Pre-adsorbed antibodies
    • Pre-adsorbed Alexa Fluor® secondaries
      • Pre-adsorbed HRP secondaries
        • Pre-adsorbed biotin secondaries
            • Fluorescent cell imaging
              • Direct vs. indirect immunofluorescence
                • Fluorochrome chart
                    • Secondary antibodies
                      • Secondary antibody selection guide
                        • Alexa Fluor® conjugated secondary antibodies
                          • Fragment antibodies
                            • HRP conjugated secondary antibodies
                              • IRDye® conjugated secondary antibodies

                                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.

                                Find the right pre-adsorbed secondary antibody

                                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.

                                ConjugateProduct name
                                Alexa Fluor® 405 

                                Anti-rabbit Alexa Fluor® 405 (IgG H&L) preadsorbed (ab175648)

                                Anti-rat Alexa Fluor® 405(IgG H&L) preadsorbed (ab175670)

                                Anti-goat Alexa Fluor® 405 (IgG H&L) preadsorbed (ab175665)

                                Anti-chicken Alexa Fluor® 405(IgY H&L) preadsorbed (ab175675)

                                Alexa Fluor® 488

                                Anti-mouse Alexa Fluor® 488 (IgG H&L) preadsorbed (ab150117)
                                Anti-rabbit Alexa Fluor® 488 (IgG H&L) preadsorbed (ab150081)
                                Anti-rat Alexa Fluor® 488 (IgG H&L) preadsorbed (ab150153)
                                Anti-goat Alexa Fluor® 488 (IgG H&L) preadsorbed (ab150133)
                                Anti-chicken Alexa Fluor® 488 (IgY H&L) preadsorbed (ab150173)
                                Alexa Fluor® 555Anti-mouse Alexa Fluor® 555 (IgG H&L) (ab150110)
                                Anti-rabbit Alexa Fluor® 555 (IgG H&L) (ab150086)
                                Anti-rat Alexa Fluor® 555 (IgG H&L) (ab150154)
                                Anti-goat Alexa Fluor® 555 (IgG H&L) (ab150134)
                                Anti-chicken Alexa Fluor® 555 (IgY H&L) (ab150174)
                                Alexa Fluor® 594Anti-mouse Alexa Fluor® 594 (IgG H&L) (ab150120)
                                Anti-rabbit Alexa Fluor® 594 (IgG H&L) (ab150064)
                                Anti-rat Alexa Fluor® 594 (IgG H&L) (ab150156)
                                Anti-goat Alexa Fluor® 594 (IgG H&L) (ab150136)
                                Anti-chicken Alexa Fluor® 594 (IgY H&L) (ab150176)
                                Alexa Fluor® 647Anti-mouse Alexa Fluor® 647 (IgG H&L) (ab150119)
                                Anti-rabbit Alexa Fluor® 647 (IgG H&L) (ab150083)
                                Anti-rat Alexa Fluor® 647 (IgG H&L) (ab150155)
                                Anti-goat Alexa Fluor® 647 (IgG H&L) (ab150135)
                                Anti-chicken Alexa Fluor® 647 (IgY H&L) (ab150175)


                                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 NameApplications
                                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 NameApplications
                                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. 

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