Primary Antibody Conjugates

Conjugated primary antibodies are an excellent alternative to using a secondary antibody for detection of your target protein. A conjugated primary antibody is created by pre-attaching (conjugating) a tag of your choice (fluorophore, enzyme, or protein), to a primary antibody, allowing for visualization of your target protein without the need to purchase a costly secondary antibody.

Conjugated primary antibodies can easily replace your previous primary/secondary antibody pairs in the following applications*:

  • Western blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Immunocytochemistry (ICC), Flow Cytometry (FCM), Fluorescence-activated cell sorting (FACS), Enzyme-Linked Immunosorbent Assay (ELISA), Förster Resonance Energy Transfer (FRET)

 

Choose from one of the following 17 different conjugate tags:

Protein and Enzymes:

Fluorophores:

Label Excitation Max. Emission Max. Extinction Coefficient Substitute for: Emission
ATTO 390
390
479
2.4×104
494
520
7.3×104
Alexa Fluor® 488
501
523
9.0×104
Alexa Fluor® 488
FITC
565
575
2.0×106
TRITC
563
592
1.2×105
Cy®3.5
ROXTM
Alexa Fluor® 546
Alexa Fluor® 555
Alexa Fluor® 568
535
627
N/A
601
627
1.2×105
Alexa Fluor® 594
Texas Red®
629
657
1.3×105
Alexa Fluor® 633
650
660
7.0×105 Cy®5
Alexa Fluor® 647
Alexa Fluor® 660
482
677
1.96 x 106
663
684
1.25×105
Cy®5
Alexa Fluor® 647
Alexa Fluor® 660
680
700
1.25×105
Cy®5.5
Alexa Fluor® 680
700
719
1.2×105
Cy®5.5
Alexa Fluor® 700

 

Protein and Enzymes


 

AP (Alkaline Phosphatase)

Properties:

  • Broad enzymatic activity for phosphate esters of alcohols, amines, pyrophosphate, and phenols
  • Commonly used to dephosphorylate the 5’-termini of DNA and RNA to prevent self-ligation
  • Catalyzes the conversion of:
    • Chromogenic substrates (e.g. pNPP, naphthol AS-TR phosphate, BCIP) into coloured products
    • Fluorogenic substrates (e.g. 4-methylumbelliferyl phosphate) into fluorescent products
  • Molecular weight: 140 kDa
  • Applications: Western blot, immunohistochemistry, and ELISA

AP Datasheet


HRP (Horseradish peroxidase)

Properties:

  • Enzymatic activity is used to amplify weak signals and increase visibility of a target
  • Readily combines with hydrogen peroxide (H2O2) to form HRP-H2O2 complex which can oxidize various hydrogen donors
  • Catalyzes the conversion of:
    • Chromogenic substrates (e.g. TMB, DAB, ABTS) into coloured products
    • Chemiluminescent substrates (e.g. luminol and isoluminol) into light emitting products via enhanced chemiluminescence (ECL)
    • Fluorogenic substrates (e.g. tyramine, homovanillic acid, and 4-hydroxyphenyl acetic acid) into fluorescent products
  • High turnover rate enables rapid generation of a strong signal
  • 44 kDa glycoprotein
  • Extinction coefficient: 100 (403 nm)
  • Applications: Western blot, immunohistochemistry, and ELISA

HRP Datasheet


BiotinBiotin Conjugate Structure

Properties:

  • Binds tetrameric avidin proteins including Streptavidin and neuravidin with very high affinity
  • Molar mass: 244.31 g/mol
  • Formula: C10H16N2O3S
  • Applications: Western blot, immunohistochemistry, and ELISA

Biotin Datasheet


Streptavidin

Properties:

  • Homo-tetrameric protein purified from Streptomyces avidinii which binds four biotin molecules with extremely high affinity
  • Molecular weight: 53 kDa
  • Formula: C10H16N2O3S
  • Applications: Western blot, immunohistochemistry, and ELISA

Streptavidin Datasheet

Fluorophores


Fluorophores, also known as fluorochromes or fluorescent dyes, are chemicals that are excited by light at a specific wavelength and then emit light (fluoresce) at another wavelength.

Key features of fluorophores:

Maximum Excitation Wavelength (λex): The peak in the excitation spectra measured in nanometers (nm).

Maximum emission Wavelength em): The peak in the emission spectra measured in nanometers (nm).

Stokes Shift: The difference in the maximum wavelength peaks between the emission and excitation spectra, measured in nanometers.

Extinction Coefficient (εmax): The capacity for the fluorophore to absorb light at a specific wavelength. Usually measured at the maximum excitation wavelength with the units M−1cm−1.

Fluorescence Quantum yield (Φf): The number of photons emitted per absorbed photon. This number will range between 0 and 1. A high quantum yield is important.

Fluorescence Decay Time (τfl): The time interval after which the number of excited fluorophore molecules decreases to 1/e (approx. 37%), usually measured in nanoseconds.

Brightness: The fluorescence output per fluorophore measured in with the units M−1cm−1. Calculated as the product of the extinction coefficient (at the relevant excitation wavelength) and the fluorescence quantum yield divided by 1000.

 ATTO Fluorophore Emission Spectrum

All ATTO fluorescent dyes are characterized by:

  • Strong absorption (high extinction coefficient)
  • High fluorescence quantum yield
  • High photostability
  ATTO 390
Overview:

  • High fluorescence yield
  • Large Stokes-shift (89 nm)
  • Good photostability
  • Moderately hydrophilic
  • Good solubility in polar solvents
  • Coumarin derivate, uncharged
  • Low molar mass: 343.42 g/mol 

ATTO 390 Datasheet

ATTO 390 Fluorescent Dye Excitation and Emission Spectra Optical Properties:

λex = 390 nm
λem = 479 nm
εmax = 2.4×104
Φf = 0.90
τfl = 5.0 ns
Brightness = 21.6
Laser = 365 or 405 nm

  FITC (Fluorescein)
Overview:

  • Excellent fluorescence quantum yield
  • High rate of photobleaching
  • Good solubility in water
  • Broad emission spectrum
  • pH dependent spectra
  • Molecular formula: C20H12O5
  • Molar mass: 332.3 g/mol

FITC Datasheet

FITC Fluorescein Fluorophore Excitation and Emission Spectra Optical Properties:

λex = 494 nm
λem = 520 nm
εmax = 7.3×104
Φf = 0.92
τfl = 5.0 ns
Brightness = 67.2
Laser = 488 nm
Filter set = FITC

  ATTO 488
Overview:

  • High fluorescence yield
  • High photostability
  • Very hydrophilic
  • Excellent solubility in water
  • Very little aggregation
  • New dye with net charge of -1
  • Molar Mass: 804 g/mol 

ATTO 488 Datasheet

  ATTO 488 Fluorophore Excitation and Emission Spectra Optical Properties:

λex = 501 nm
λem = 523 nm
εmax = 9.0×104
Φf = 0.80
τfl = 4.1 ns
Brightness = 72
Laser = 488 nm
Filter set = FITC

  R-PE (R-Phycoerythrin)
Overview:

  • Broad excitation spectrum
  • High quantum yield
  • Photostable
  • Member of the phycobiliprotein family
  • Isolated from red algae
  • Excellent solubility in water
  • Molecular Weight: 250 kDa

R-PE Datasheet

 R-PE Fluorophore Excitation and Emission Spectra Optical Properties:

λex = 565 nm
λem = 575 nm
εmax = 2.0×106
Φf = 0.84
Brightness = 1.68 x 103
Laser = 488 to 561 nm
Filter set = TRITC

 ATTO 565
Overview:

  • High fluorescence yield
  • High thermal and photostability
  • Good solubility in polar solvents
  • Excellent solubility in water
  • Very little aggregation
  • Rhodamine dye derivative
  • Molar Mass: 611 g/mol

ATTO 565 Datasheet

 ATTO 565 Fluorophore Excitation and Emission Spectra Optical Properties:

λex = 563 nm
λem = 592 nm
εmax = 1.2×105
Φf = 0.90
τfl = 3.4 ns
Brightness = 108
Laser = 532 nm
Filter set = TRITC

 PE/ATTO 594
PE/ATTO 594 is a tandem conjugate, where PE is excited at 535 nm and transfers energy to ATTO 594 via FRET (fluorescence resonance energy transfer), which emits at 627 nm.
Overview:

  • High fluorescence yield
  • High photostability
  • Very hydrophilic
  • Excellent solubility in water
  • Very little aggregation

PE/ATTO 594 Datasheet

PE-ATTO 594 Fluorophore Conjugate Excitation and Emission Spectra Optical Properties:

λex = 535 nm
λem = 627 nm
Laser = 488 to 561 nm

 ATTO 594
Overview:

  • High fluorescence yield
  • High photostability
  • Very hydrophilic
  • Excellent solubility in water
  • Very little aggregation
  • New dye with net charge of -1
  • Molar Mass: 1137 g/mol

ATTO 594 Datasheet

 ATTO 594 Fluorophore Excitation and Emission Spectrum Optical Properties:

λex = 601 nm
λem = 627 nm
εmax = 1.2×105
Φf = 0.85
τfl = 3.5 ns
Brightness = 102
Laser = 594 nm
Filter set = Texas Red®

 ATTO 633
Overview:

  • High fluorescence yield
  • High thermal and photostability
  • Moderately hydrophilic
  • Good solubility in polar solvents
  • Stable at pH 4 – 11
  • Cationic dye, perchlorate salt
  • Molar Mass: 652.2 g/mol

ATTO 633 Datasheet

 ATTO 633 Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 629 nm
λem = 657 nm
εmax = 1.3×105
Φf = 0.64
τfl = 3.2 ns
Brightness = 83.2
Laser = 633 nm
Filter set = Cy®5

APC (Allophycocyanin)
Overview:

  • High quantum yield
  • Large phycobiliprotein
  • 6 chromophores per molecule
  • Isolated from red algae
  • Molecular Weight: 105 kDa 

APC Datasheet

 APC Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 650 nm
λem = 660 nm
εmax = 7.0×105
Φf = 0.68
Brightness = 476
Laser = 594 or 633 nm
Filter set = Cy®5

 PerCP 
Overview:

  • Peridinin-Chlorophyll-Protein Complex
  • Small phycobiliprotein
  • Isolated from red algae
  • Large stokes shift (195 nm)
  • Molecular Weight: 35 kDa

PerCP Datasheet

 PerCP Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 482 nm
λem = 677 nm
εmax = 1.96 x 106
Laser = 488 nm

 ATTO 655
Overview:

  • High fluorescence yield
  • High thermal and photostability
  • Excellent ozone resistance
  • Quenched by electron donors
  • Very hydrophilic
  • Good solubility in polar solvents
  • Zwitterionic dye
  • Molar Mass: 634 g/mol

ATTO 655 Datasheet

ATTO 655 Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 663 nm
λem = 684 nm
εmax = 1.25×105
Φf = 0.30
τfl = 1.8 ns
Brightness = 37.5
Laser = 633 – 647 nm
Filter set = Cy®5

 ATTO 680
Overview:

  • High fluorescence yield
  • Excellent thermal and photostability
  • Quenched by electron donors
  • Very hydrophilic
  • Good solubility in polar solvents
  • Zwitterionic dye
  • Molar Mass: 631 g/mol

ATTO 680 Datasheet

 ATTO 680 Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 680 nm
λem = 700 nm
εmax = 1.25×105
Φf = 0.30
τfl = 1.7 ns
Brightness = 37.5
Laser = 633 – 676 nm
Filter set = Cy®5.5

 ATTO 700
Overview:

  • High fluorescence yield
  • Excellent thermal and photostability
  • Quenched by electron donors
  • Very hydrophilic
  • Good solubility in polar solvents
  • Zwitterionic dye
  • Molar Mass: 575 g/mol

ATTO 700 Datasheet

 ATTO 700 Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 700 nm
λem = 719 nm
εmax = 1.25×105
Φf = 0.25
τfl = 1.6 ns
Brightness = 31.3
Laser = 676 nm
Filter set = Cy®5.5

 


*The applications listed above are a general list indicating which applications conjugated antibodies can be used in. It does not guarantee that any antibody-conjugate combination has been tested for use in the application.

**Excludes lyophilized antibodies. Please see product pages for conjugate availability.

Please note: It is possible that the conjugate tag may bind in the paratope of the antibody, thereby limiting binding of the antibody to the antigen. Although this is unlikely, it could affect the ability of the antibody to bind to the antigen in various species and applications. As we cannot control the binding of the conjugate to the antibody, there is no way to confirm or guarantee the location of the antibody tag.

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