Secondary Antibody Handbook

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Secondary Antibody Handbook

This secondary antibody selection handbook will provide an introduction to antibody structure, secondary antibody nomenclature and formats, and technical information to help you choose the best secondary antibody for your western blot, immunohistochemistry, or flow cytometry experiments.

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Description / Type


       Notes


Host Species


The species recognized by the secondary antibody.


Types
  • Goat
  • Donkey
  • Rabbit
  • Mouse



  • All secondary antibodies should be derived from the same host species when possible.

  • Block with serum from the same host species as the secondary antibody to reduce non-specific binding.


Format


The structure of the secondary antibody.


Types
  • F(ab)
  • F(ab’)
  • F(ab’)
  • Whole Ig

  • To stain samples with high Fc receptor expression (e.g. lymph node, spleen, peripheral blood, etc) a Fab fragment secondary antibody is recommended

  • A Fab fragment secondary antibody can be used to double label with multiple primary antibodies from the same host species.

  • The smaller size of Fab fragment antibodies can enhance tissue penetration and improve sensitivity in IHC.




Target Ig Class


The immunoglobulin class recognized by the secondary antibody.


Types
  • Classes: IgA, IgD, IgE, IgG, IgM
  • Human Subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2
  • Mouse Subclasses: IgG1, IgG2a, IgG2b, IgG3
  • Light Chains: Kappa and Alpha



  • The secondary antibody should be directed against the class or subclass of the primary antibody.

  • Subclass specific secondary antibodies are used in multiple labeling experiment when multiple primary antibodies derived from unique subclasses are used


Specificity


The region of the Ig or primary antibody recognized by the secondary antibody.


Types
  • H+L
  • F(ab)
  • Fc
  • Heavy chain
  • Light chain

  • A secondary antibody targeting the light chain (kappa or lambda) will bind to any class. Light chains are shared between classes.


Conjugates


The enzymatic or fluorescent label conjugated to the secondary antibody.


Types
  • Fluorescent
  • HRP, AP
  • Biotin



  • The choice of conjugate is application dependent.

  • Biotin conjugates allow signal amplification.

  • Fluorescent conjugates allow easier multiplexing.