Jackson Immuno Research Inc.
specializing in secondary antibodies and conjugates

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Secondary Antibodies

For VHH Discovery

Anti-Alpaca IgG, Subclass and VHH domain secondaries
  • Ideally time PBMC harvest
  • Enhance detection of VHH antibodies
  • Test for VHH expression and binding activity

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Whole IgG Affinity-Purified Secondary Antibodies

"I have used a wide variety of secondaries and Jackson ImmunoResearch has consistently been the best. The fluorophores are bright and stable and their selective (x reactivity removed) secondaries have always shown species specificity in multiple labeling."

Janet Duerr, Ohio University

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Reporter Molecules - Fluorophores

Technical Service e-mail
tech@jacksonimmuno.com

Fluorophore Selection

Fluorescent probes or fluorophores (fluorescent dyes or proteins) are coupled to a secondary antibody or streptavidin to allow visualization of an analyte. Each fluorophore has its own spectral characteristics, with excitation and emission spectra particular to the molecule. Fluorescence facilitates the simultaneous detection of multiple analytes for a number of techniques including flow cytometry, fluorescence microscopy (epifluorescence, confocal, multiphoton and super-resolution techniques), Western blotting and ELISA.

The choice of fluorescent probe depends on a number of experimental variables, described below. There is also a Spectra Viewer Tool to assist in experimental design.

Further information about each of the fluorescent dyes offered is available on the Fluorophore Characteristics page.


Technique

Selection of a fluorophore depends on the intended application. Sample specifics influence the choice, as they may accommodate the use of particular fluorophores, and preparation may alter the way a fluorophore behaves. The following table lists some of the considerations which are pertinent to the choice of a fluorophore for a particular technique.

Technique Considerations
Flow cytometry Surface staining accommodates the use of larger and brighter fluorescent conjugates such as the fluorescent proteins (R-PE, APC and PerCP) or Brilliant Violet™ dyes. Smaller fluorescent conjugates can be used for both surface and intracellular staining.
IHC microscopy
  • Sample penetration requirements
  • Sample autofluorescence
  • Polar (aqueous) or non-polar (plastic) mounting media
  • pH
  • Analyte abundance - choose a brighter dye for weakly expressing targets
IHC multiple labeling
  • Spectral overlap of fluorophores
  • Filter sets available
  • Analyte abundance - choose a brighter dye for the least abundant analyte
Super-resolution microscopy (STED and STORM)
  • High emission at the STED laser wavelength - achieving high saturation
  • Photostability
  • Brightness
  • Photoswitching
Western and dot blotting
  • Spectral overlap
  • Far-red and infrared fluorescent dyes for high sensitivity

Table 1: Considerations for conjugate selection.


Instrument capabilities

Consider excitation capabilities (lamp, lasers or LEDs determine excitation wavelengths) and the number of channels and filters available. Also consider the detection system, duration of data collection and post assay analysis.


Sensitivity required

The detection level of any fluorophore-antibody conjugate depends on brightness and photostability of the dye; antibody activity, specificity and cross-reactivity; and the optimal dye:antibody ratio (moles of dye per mole of antibody). These parameters have been researched for each of JIR’s dye conjugates to optimize the level of antibody detection and minimize background. The larger phycobiliproteins have high quantum yields (are very bright), but are limited to surface applications. Detection of poorly expressed analytes is enhanced by choosing a brighter fluorophore. For example, Alexa Fluor® 488 is brighter than FITC.


Experimental sample

Consider autofluorescence of the sample and possible expression of recombinant fluorescently tagged proteins. Choose fluorophores whose spectra do not overlap with endogenous fluorescence.


Degree of color separation required

For multiple labeling protocols, the dye panel choices will be constrained by instrumentation and sample specifics as described above. To achieve good color separation, choose fluorophores with minimal spectral overlap. The Spectra Viewer below can help build dye panels specific to any instrument.

Read more information on IHC multiple labeling


Spectra Viewer

Fluorescent probes or fluorophores (fluorescent dyes or proteins) are coupled to a secondary antibody or streptavidin to allow visualization of an analyte. Each fluorophore has its own spectral characteristics, with excitation and emission spectra particular to the molecule. Use the spectral viewer to build dye panels and compare the suitability of dyes for your application. Four dyes typically recommended for multiple labeling are pre-loaded, these can be changed as required.

Read our blog about selecting fluorophores using the Spectra Viewer


For further assistance please contact our technical support team who will be delighted to assist you.

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