Nano-Secondaries

HeLa cells stably expressing Tubulin-GFP at near-endogenous level were immunostained with rabbit anti-GFP PABG1 antibody 1:1,000 and alpaca anti-rabbit IgG, recombinant VHH, Alexa Fluor® 647 1:1,000 (magenta), nuclei were detected with H2B-RFP signal (cyan). Scale bar, 20 µm.
Confocal images were acquired with a Leica TCS SP8 microscope, 100x oil objective, and deconvolved with Huygens Professional (SVI). Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

Nano-Secondaries decreases the distance between fluorophores and epitope. Comparison of a primary antibody that is 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.

Standard sequential antibody incubation immunostaining (1’,2’) vs. simultaneous one-step immunostaining (1’+2’): HeLa cells were immunostained with rabbit anti-βActin, anti-Histone H2A, anti-Ki67, anti-LaminB1 (left to right) and alpaca anti-rabbit IgG, recombinant VHH, Alexa Fluor® 647 (1:1,000). Primary antibodies and secondary alpaca anti-rabbit IgG, recombinant VHH, Alexa Fluor® 647 were applied either sequentially following a standard immunostaining protocol (upper row) or simultaneously as one-step immunostaining (lower row), indicating the feasibility of one-step staining. Scale bar, 20 µm. Epifluorescence images were acquired with CellInsight CX7 HCS microscope, 20X objective.

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 antibodies, alpacas possess also single domain antibodies (sdAbs). sdAbs contain only heavy chains and are devoid of light chains. The antigen binding domain of a sdAb 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, recombinant 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 IgG1VHH 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.

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/ V_HHs 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

Nano-Secondaries
Alpaca anti-rabbit IgG, recombinant V_HH
Alpaca anti-mouse IgG1, recombinant V_HH
Alpaca anti-mouse IgG2b, recombinant V_HH
Alpaca anti-mouse IgG3, recombinant V_HH

Product Listing
Applications

Alpaca anti-mouse IgG1, recombinant VHH

ORDER Alpaca anti-mouse IgG1, recombinant VHH

IF SRM WB ELISA

Code	Description	Size	Price	Qty
Code sms1AF647-1-10	Description Alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 647	Size 10 µL	Price $ 100	Qty + +
Code sms1AF647-1-100	Description Alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 647	Size 100 µL	Price $ 365	Qty + +

Alpaca anti-mouse IgG2b, recombinant VHH

ORDER Alpaca anti-mouse IgG2b, recombinant VHH

IF SRM WB ELISA

Code	Description	Size	Price	Qty
Code sms2bAF647-1-10	Description Alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 647	Size 10 µL	Price $ 100	Qty + +
Code sms2bAF647-1-100	Description Alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 647	Size 100 µL	Price $ 365	Qty + +

Alpaca anti-mouse IgG3, recombinant VHH

ORDER Alpaca anti-mouse IgG3, recombinant VHH

IF SRM WB ELISA

Code	Description	Size	Price	Qty
Code sms3AF647-1-10	Description Alpaca anti-mouse IgG3, Fab-specific recombinant VHH, Alexa Fluor® 647	Size 10 µL	Price $ 100	Qty + +
Code sms3AF647-1-10	Description Alpaca anti-mouse IgG3, Fab-specific recombinant VHH, Alexa Fluor® 647	Size 100 µL	Price $ 365	Qty + +

Alpaca anti-rabbit IgG, recombinant VHH

ORDER Alpaca anti-rabbit IgG, recombinant VHH

IF SRM WB ELISA

Code	Description	Size	Price	Qty
Code srbAF488-1-10	Description Alpaca anti-rabbit IgG, recombinant VHH, Alexa Fluor® 488	Size 10 µL	Price $ 100	Qty + +
Code srbAF488-1-100	Description Alpaca anti-rabbit IgG, recombinant VHH, Alexa Fluor® 488	Size 100 µL	Price $ 365	Qty + +
Code srbAF568-1-10	Description Alpaca anti-rabbit IgG, recombinant VHH, Alexa Fluor® 568	Size 10 µL	Price $ 100	Qty + +
Code srbAF568-1-100	Description Alpaca anti-rabbit IgG, recombinant VHH, Alexa Fluor® 568	Size 100 µL	Price $ 365	Qty + +
Code srbAF647-1-10	Description Alpaca anti-rabbit IgG, recombinant VHH, Alexa Fluor® 647	Size 10 µL	Price $ 100	Qty + +
Code srbAF647-1-100	Description Alpaca anti-rabbit IgG, recombinant VHH, Alexa Fluor® 647	Size 100 µL	Price $ 365	Qty + +

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 V_HH respective Nanobody
are fully characterized and validated
are recombinantly produced, high lot-to-lot consistency

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

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