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Nano-Secondaries

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HeLa cells stably expressing Tubulin-GFP at near-endogenous level were immunostained with rabbit anti-GFP PABG1 antibody and alpaca anti-rabbit IgG VHH Alexa Fluor® 647 (magenta). Nuclei were detected with H2B-RFP and RFP-Booster Atto594 (cyan). Scale bar, 10 µm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
Nano-Secondaries in confocal and super resolution microscopy: HeLa cells were immunostained with rabbit anti-Lamin B1 antibodies and alpaca anti-rabbit IgG VHH Alexa Fluor® 568 (1:1,000). Confocal and gated STED images were acquired with a Leica TCS SP8 STED 3X microscope, pulsed depletion with a 775 nm laser. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
Nano-Secondaries decrease the distance between fluorophores and epitope. Comparison of a primary antibody stained with Nano-Secondaries (left) and polyclonal secondaries (right): Formation of a small complex of 2 Fab- and 2 Fc-specific Nano-Secondaries (green) & primary antibody (grey) to label the epitope (magenta). Formation of a large complex of multiple polyclonal secondaries (green) & primary antibody to label the epitope.
Vimentin in HeLa cells with Leica GSDIM system. The cells were stained with monoclonal anti-Vimentin mouse IgG1 antibody and alpaca anti-mouse IgG1 VHH Alexa Fluor® 488. The image is a courtesy of Dr. Leila Nahidiazar, Dr. Jop Kind, and Prof. Kees Jalink.
One-step immunostaining is the simultaneous incubation of primary antibody and Nano-Secondary. This method reduces incubation and hands-on time. Simultaneous incubation also supports multiplexing, tissue penetration, and cell staining for flow cytometry.
Next to conventional IgG antibodies, alpacas also possess heavy chain only IgGs. These antibodies lack the CH1 domain of the heavy chain and are devoid of any light chain. The antigen binding domain of the heavy chain only IgGs is called VHH or Nanobody. Nano-Secondaries are IgG-specific Nanobodies that are coupled to Alexa Fluor dyes.
One-step immunostaining vs. sequential immunostaining of HeLa cells. Primary antibodies + secondary alpaca anti-rabbit IgG VHH Alexa Fluor® 647. Scale bar, 20 μm.
Multiplexed immunostaining of HeLa cells with 3 subclass-specific alpaca anti-mouse Nano-Secondaries. Grey: Mouse IgG1 anti-Vimentin + alpaca anti-mouse IgG1 VHH Alexa Fluor® 647. Green: Mouse IgG2b anti-Lamin + alpaca anti-mouse IgG2b VHH Alexa Fluor® 488. Red: Mouse IgG3 anti-MOT + alpaca anti-mouse IgG3 VHH Alexa Fluor® 568. Scale bar, 10 µm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
Immunostaining with Nano-Secondaries for flow cytometry. Jurkat CD3+ and Jurkat CD3- cell lines were mixed and immunostained live with anti-CD3 mouse IgG1 + alpaca anti-mouse IgG1 VHH Alexa Fluor® 647 (1:600). CD3- cells were pre-stained with Calcein. Two cell populations can be clearly distinguished on the dot-plot: Alexa Fluor® 647-positive Calcein-negative cells and Alexa Fluor® 647-negative Calcein-positive cells.
ChromoTek's alpaca anti-rabbit IgG VHH is highly specific and binds only rabbit gamma immunoglobulins (IgG). In contrast, competitor A’s goat anti-rabbit IgG antibody shows unwanted cross-reactivity to mouse IgGs (red square) despite its pre-adsorption against mouse serum.

Description
Nano-Secondaries® are a novel class of secondary antibodies for higher resolution & cleaner images. They consist of Nanobodies/ VHHs that bind to primary antibodies with high affinity in a species and subtype specific manner. Nano-Secondaries are conjugated to Alexa Fluor® dyes.

The next level of secondary antibodies
Higher resolution in immunofluorescence
Fast one-step immunostaining
No Cross-reactivity to IgG of other frequently used species and subtypes

Applications
Immunofluorescence
Super-resolution microscopy
Western blotting
Flow cytometry

Nano-Secondaries
Nano-Secondary alpaca anti-human IgG
Nano-Secondary alpaca anti-mouse IgG1
Nano-Secondary alpaca anti-mouse IgG2b
Nano-Secondary alpaca anti-mouse IgG3
Nano-Secondary alpaca anti-rabbit IgG

Brochure
Nano-Secondaries product brochure (PDF)
 

Nano-Secondary alpaca anti-human IgG

PRICE Nano-Secondary alpaca anti-human IgG
IF SRM WB ELISA
x
Code Description Size Price Qty
Code srbAF488-1-10 Description Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 488 [CTK0101, CTK0102] Size 10 µL Price $ 100
Qty +
Code srbAF488-1-100 Description Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 488 [CTK0101, CTK0102] Size 100 µL Price $ 365
Qty +
Code srbAF568-1-10 Description Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 568 [CTK0101, CTK0102] Size 10 µL Price $ 100
Qty +
Code srbAF568-1-100 Description Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 568 [CTK0101, CTK0102] Size 100 µL Price $ 365
Qty +
Code srbAF647-1-10 Description Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 647 [CTK0101, CTK0102] Size 10 µL Price $ 100
Qty +
Code srbAF647-1-100 Description Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 647 [CTK0101, CTK0102] Size 100 µL Price $ 365
Qty +
Code srbGCys2-1-500 Description Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, for 2x Cys conjugation [CTK0101] Size 500 μg Price Please inquire

Nano-Secondary alpaca anti-mouse IgG1

PRICE Nano-Secondary alpaca anti-mouse IgG1
IF SRM WB ELISA
x
Code Description Size Price Qty
Code sms1AF488-1-10 Description Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 488 [CTK0103, CTK0104] Size 10 µL Price $ 100
Qty +
Code sms1AF488-1-100 Description Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 488 [CTK0103, CTK0104] Size 100 µL Price $ 365
Qty +
Code sms1AF568-1-10 Description Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 568 [CTK0103, CTK0104] Size 10 µL Price $ 100
Qty +
Code sms1AF568-1-100 Description Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 568 [CTK0103, CTK0104] Size 100 µL Price $ 365
Qty +
Code sms1AF647-1-10 Description Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 647 [CTK0103, CTK0104] Size 10 µL Price $ 100
Qty +
Code sms1AF647-1-100 Description Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 647 [CTK0103, CTK0104] Size 100 µL Price $ 365
Qty +
Code smsG1Cys2-1-500 Description Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, for 2x Cys conjugation [CTK0103] Size 500 μg Price Please inquire
Code smsG1Cys2-2-500 Description Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, for 2x Cys conjugation [CTK0104] Size 500 μg Price Please inquire

Nano-Secondary alpaca anti-mouse IgG2b

PRICE Nano-Secondary alpaca anti-mouse IgG2b
IF SRM WB ELISA
x
Code Description Size Price Qty
Code sms2bAF488-1-10 Description Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, Alexa Fluor® 488 [CTK0105, CTK0106] Size 10 µL Price $ 100
Qty +
Code sms2bAF488-1-100 Description Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, Alexa Fluor® 488 [CTK0105, CTK0106] Size 100 µL Price $ 365
Qty +
Code sms2bAF568-1-10 Description Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, Alexa Fluor® 568 [CTK0105, CTK0106] Size 10 µL Price $ 100
Qty +
Code sms2bAF568-1-100 Description Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, Alexa Fluor® 568 [CTK0105, CTK0106] Size 100 µL Price $ 365
Qty +
Code sms2bAF647-1-10 Description Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, Alexa Fluor® 647 [CTK0105, CTK0106] Size 10 µL Price $ 100
Qty +
Code sms2bAF647-1-100 Description Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, Alexa Fluor® 647 [CTK0105, CTK0106] Size 100 µL Price $ 365
Qty +
Code smsG2bCys2-1-500 Description Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, for 2x Cys conjugation [CTK0105] Size 500 μg Price Please inquire
Code smsG2bCys2-2-500 Description Nano-Secondary® alpaca anti-mouse IgG2b, recombinant VHH, for 2x Cys conjugation [CTK0106] Size 500 μg Price Please inquire

Nano-Secondary alpaca anti-mouse IgG3

PRICE Nano-Secondary alpaca anti-mouse IgG3
IF SRM WB ELISA
x
Code Description Size Price Qty
Code sms3AF647-1-10 Description Nano-Secondary® alpaca anti-mouse IgG3, recombinant VHH, Alexa Fluor® 647 [CTK0107] Size 10 µL Price $ 100
Qty +
Code sms3AF647-1-100 Description Nano-Secondary® alpaca anti-mouse IgG3, recombinant VHH, Alexa Fluor® 647 [CTK0107] Size 100 µL Price $ 365
Qty +

Nano-Secondary alpaca anti-rabbit IgG

PRICE Nano-Secondary alpaca anti-rabbit IgG
IF SRM WB ELISA
x
Code Description Size Price Qty
Code srbAF488-1-10 Description Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 488 [CTK0101, CTK0102] Size 10 µL Price $ 100
Qty +
Code srbAF488-1-100 Description Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 488 [CTK0101, CTK0102] Size 100 µL Price $ 365
Qty +
Code srbAF568-1-10 Description Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 568 [CTK0101, CTK0102] Size 10 µL Price $ 100
Qty +
Code srbAF568-1-100 Description Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 568 [CTK0101, CTK0102] Size 100 µL Price $ 365
Qty +
Code srbAF647-1-10 Description Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 647 [CTK0101, CTK0102] Size 10 µL Price $ 100
Qty +
Code srbAF647-1-100 Description Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 647 [CTK0101, CTK0102] Size 100 µL Price $ 365
Qty +
Code srbGCys2-1-500 Description Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, for 2x Cys conjugation [CTK0101] Size 500 μg Price Please inquire
Code srbGCys2-2-500 Description Nano-Secondary® alpaca anti-rabbit IgG, recombinant VHH, for 2x Cys conjugation [CTK0102] Size 500 μg Price Please inquire

One-step immunostaining protocol saves time

ChromoTek’s Nano-Secondaries consist of monovalent Nanobodies with very high affinity. Hence, they enable one-step immunostaining: the simultaneous incubation of primary antibody and Nano-Secondary.
This method saves incubation time and washing steps. Simultaneous incubation also supports: 

  • multiplexing, 
  • tissue penetration, and
  • cell staining for flow cytometry.

Of course, Nano-Secondaries can be used according to standard immunostaining protocols following a sequential incubation of primary and Nano-Secondary, too. In sequential staining, you can simply substitute the Nano-Secondary for any secondary antibody.

Better imaging

  • Better tissue penetration and small epitope-label displacement for higher resolution
  • High sensitivity: detection of low expressed epitopes via primary antibody
  • High affinity reduces background

No Cross-reactivity to IgGs of other frequently used species

The cross reactivity of the Nano-Secondary has been carefully tested. It shows no cross-reactivity to IgG & serum of frequently used species. Because of their high specificity our Nano-Secondaries do not require any kind of pre-adsorption.

Precise and reproducible staining

Nano-Secondaries enable very precise and reproducible stainings. In detail, they:

  • bind in a site-specific manner to primary IgG antibodies 
  • are stoichiometrically labeled with Alexa Fluor dyes per VHH respective Nanobody
  • are fully characterized and validated
  • are recombinantly produced, high lot-to-lot consistency

Nano-secondaries are is totally animal free manufactured, though VHHs respective Nanobodies are generated from immunization of alpacas and llamas.

 

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Only for research applications, not for diagnostic or therapeutic use!