Conjugate guide | Biotin, HRP, AP and Fluorochrome

The type of conjugation is application dependent.

For enzymatic and biotin detection, e.g. in WB or ELISA, we suggest a secondary antibody conjugated to horseradish peroxidase (HRP), alkaline phosphatase (AP) or biotin. Both avidin and streptavidin bind very strongly to biotin and enable signal amplification, regardless of the host species of the antibody.

If a laser light is involved, e.g. in Flow Cytometry, ICC/IF or IHC, we suggest fluorescent detection with a secondary antibody conjugated to a fluorochrome.

View more details about secondary detection methods:

 

Detection with biotin

For amplification of signal, a two-step biotin-avidin-enzyme may be used (commonly abbreviated as "ABC" reagent). A biotinylated secondary antibody is applied first, then avidin or streptavidin conjugated to an enzyme or fluorochrome binds to multiple sites on the biotinylated secondary antibody.

Learn more about biotin binding proteins.

 

Enzymatic detection

HRP, popular for use in chemiluminescent detection systems, is less expensive, rapid and a more stable enzyme than AP. However, AP is considered more sensitive particularly when colorimetric detection is used.

Table: Guide to enzymatic detections

Enzyme

Substrate

Applications

Advantages

Drawbacks

HRP

Chromogenic, soluble (TMB, ABTS, OPD...)

ELISA

Easy to use

Light sensititive coloration

 

Chromogenic, precipitating (CN, AEC, DAB,...)

WB, SB, IHC

Easy to use

Background in blood samples and some other tissues

Staining stability lower than AP

Fluorogenic (ADHP/resorufin)

Chemiluminescent

ELISA

High sensitivity

Need a fluorescent equipment

 

Luminol

WB, SB, IHC

High sensitivity

Need radiographic equipment or light scanner

AP

Chromogenic, soluble (pNPP)

ELISA

Linear kinetic

Often more sensitive than HRP

Unstable

Chromogenic, precipitating(BCIP/NBT,...)

WB, SB, IHC

Staining stability higher than HRP

Interfere with nuclear counterstain

 

Fluorogenic (4-MUP)

ELISA, IHC

Sensitivity

Need fluorescence equipment

WB: Western Blot, SB: Southern Blot, IHC: Immunohistochemistry, ELISA: Enzyme linkied immunosorbent assay

Fluorescent detection

Due to their novel electronic configurations, fluorochromes have a unique and characteristic spectra for absorption (excitation) and emission.

A single dye is excited at a particular wavelength and emits a photon at another wavelength.

A tandem dye consists of a donor and acceptor fluorochrome molecule, placed in close proximity, allowing for energy transfer between the two. The tandem dye is excited at the excitation wavelength of the acceptor molecule and emits a photon at the emission wavelength of the donor molecule.

Guide to fluorochromes 

PDF Download the guide to fluorochromes

  Learn more about fluorochromes and how fluorescent dyes work.

The guide is also available in our 'Understanding secondary antibodies' card. PDF Download the entire card (8 MB) or email us to request your free copy today!


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