Secondary Antibodies

Choosing the Right Secondary Antibody

The right secondary antibody depends on three factors: your primary antibody's host species, your detection method, and whether you need to detect multiple targets simultaneously.

Three Types, Three Strengths

Multi-rAb recombinant secondaries are Proteintech's most advanced option. They combine the sensitivity of polyclonal antibodies — recognizing multiple epitopes on the primary antibody's Fc region — with the specificity and lot-to-lot consistency of recombinants. They show minimal cross-reactivity with off-target species, making them suitable for multiplex experiments.

Nano-Secondary reagents are single-domain VHH antibodies derived from alpaca, approximately one-tenth the size of conventional antibodies. Their small size gives them two advantages: better tissue penetration in thick specimens and higher resolution in super-resolution microscopy. They are recombinantly produced with guaranteed batch consistency. Use Nano-Secondaries when imaging resolution or tissue penetration is your priority.

Traditional polyclonal secondaries offer the widest selection of conjugates and species specificities at the most accessible price point. They provide strong signal amplification and work reliably across standard applications. They remain a solid choice for routine Western blotting and standard immunoassays where lot-to-lot variation is less critical.

Selecting a Conjugate

Detection Method	Conjugate	Best For
Western Blot	HRP, CoraLite® Plus (for fluorescent WB)	Chemiluminescent detection — the standard for WB
Immunofluorescence	CoraLite® Plus	Fluorescent imaging with minimal photobleaching
Flow Cytometry	CoraLite® Plus, FITC, R-PE, APC, CoraLux	Bright fluorophores compatible with common laser lines
IHC (Chromogenic)	HRP or Biotin	DAB or ABC detection for FFPE tissue
Multiplex / Multi-color	Spectrally distinct fluorophores	Combine secondaries raised in different hosts or use direct-conjugated primaries

Avoiding Cross-Reactivity

When detecting multiple targets in the same experiment, ensure your secondary antibodies do not cross-react with each other's primary antibodies. Use primaries raised in different host species (e.g., mouse and rabbit), paired with species-specific secondaries. Alternatively, use direct-conjugated primary antibodies to bypass secondary antibody conflicts entirely. Proteintech's CoraLite® conjugated primaries are designed for this purpose.