All tags Secondary Antibodies Conjugate selection guide for secondary antibodies

Conjugate selection guide for secondary antibodies


Learn about biotin labeled antibodies, HRP and fluorescent secondary antibodies so you can choose the correct secondary antibody for your application.

Secondary antibodies can be conjugated to enzymes, biotin, and fluorescent dyes/proteins. The conjugate choice depends upon the experimental application and allows colorimetric, fluorescent  or chemiluminescent detection of primary antibodies in cell imaging, immunohistochemistry, flow cytometry and western blotting.

See table below what are the commonly used applications for different conjugated secondary antibodies such as biotinylated antibody, HRP  and Alexa Fluor® fluorescent secondary antibodies.

Immunoassay experimentSecondary antibody label

Immunocytochemistry, IHC-Fr

Alexa Fluor® DyLight®

Immunohistochemistry

Biotin , HRP or HRP-polymer

Flow cytometryAlexa Fluor®Phycoerythrin, FITC
Western blotEnzymes - HRP or AP
ELISAEnzymes -  HRP  or  biotin
See all secondary antibodies

Biotin/streptavidin

Multiple biotin molecules can be conjugated to a secondary antibody, biotinylated secondary antibody provides an amplification that makes biotin conjugated secondary antibodies suitable for detecting proteins expressed at low levels. Visualization is through interaction between biotin and streptavidin, where the streptavidin is bound to labels such as HRP or fluorescent probes. During the assay, it is normal procedure for the biotin labeled secondary antibody to be added first, sequentially followed by the streptavidin conjugated to a label.


Fluorescent labeled secondary antibodies

Fluorescent conjugates are preferred for cell imaging, flow cytometry and immunohistochemistry frozen samples (IHC-Fr). A single dye is excited at a particular wavelength and emits a photon at another wavelength, detected by fluorescent microscope. Fluorescent conjugate secondary antibodies allow signal amplification detection of primary antibodies in cells and tissues due to increase number of conjugated secondary antibodies that are able to bind to primary antibody and are able to be detected by microscopy. 

See advantages of Alexa Fluor® conjugated antibodies


Enzymes

Enzyme labels are visualized with chromogenic reactions whereby a soluble colorless substrate is converted to a water-insoluble colored compound. so enzymes like HRP and AP allow colorimetric or chemiluminescent detection of primary antibodies in application like western blot, immunohistochemistry

Commonly used enzymes:

Horseradish peroxidase (HRP) is visualized by chromogenic reactions such as diaminobenzidine (DAB) or chemiluminescence. HRP is a 44kDa glycoprotein enzyme label and is more stable than alkaline phosphatase.

HRP labeled secondary antibodies 

HRP-polymer secondaries by binding more horseradish peroxidase than standard HRP secondary antibodies allows amplification of signal ideal for those low expressing targets in IHC. See more about HRP-polymer secondary antibodies.

Alkaline phosphatase (AP) is a hydroylase enzyme and its signal is often measured through its colorimetric substrate p-nitrophenyl phosphate (pNPP). Used mostly in western blot.

AP labeled secondary antibodies

How to use fluorescent labeled secondary antibodies?

Tips for choosing a fluorescent labeled secondary antibody

  • Choose the brightest set of fluorochromes for your instrument.
  • Spectral overlap can be minimized through choosing the right fluorescent labels.
  • Make sure to combine the brightest label with the protein which has the lowest level of expression, and vice versa.
  • When using secondary antibodies, check to make sure all the primary antibodies have been raised in different species to ensure cross labeling via the secondaries is not encountered. Remember, we provide secondary antibodies against different sub-types from within the same species (mouse IgG1, IgG2, IgG2a, IgG3 etc), providing further choice.  Obviously the corresponding subtype primary must be used.
  • Pre-adsorbed secondary antibodies are generally used during multi-color analysis to ensure low cross species reactivity.
  • Sample autofluorescence is sometimes a problem and must be take in to consideration when choosing your secondary conjugates. For instance, liver sections autofluoresce in the red channel, thus when staining this tissue type labels which emit in the red channel should be avoided.


Absorption/emission spectrum and extinction coefficient

See below fluorescent conjugates absorption/emission spectrum and extinction coefficient to plan your multicolor panels.

Label

Absorption Max (nm)

Emission Max (nm)

Extinction Coefficient

Alexa Fluor® 405

402

421

35,000

Cy2

489

506

150,000

Alexa Fluor® 488

495

519

73,000

FITC

494

520

70,000

Alexa Fluor® 555

555

565

155,000

Cy3

550

570

150,000

Phycoerythrin

488

575

1,960,000

Rhodamine

544

576

80,000

Cy3.5

576

589

150,000

Alexa Fluor® 568

578

603

88,000

PE/Texas Red®

535

615

-

Texas Red®

595

615

85,000

Alexa Fluor® 594

590

617

92,000

APC

650

662

700,000

Cy5

643

667

250,000

Alexa Fluor® 647

650

668

270,000

PE/Cy7

488

670

-

Cy5.5

675

694

190,000

PE/Cy5.5

535

694

-

Alexa Fluor® 680

679

702

183,000

Alexa Fluor® 750

749

775

290,000

APC/Cy7

633

776

-

Alexa Fluor® 790

784

814

260,000


Read next advantages of using Alexa Fluor® conjugated antibodies


Alexa Fluor® and Texas Red® are registered trademarks of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.  DyLight® is a registered trademark of Thermo Fisher Scientific Inc and its subsidiaries. 

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