Nano-Secondary alpaca anti-rabbit IgG

HeLa cells stably expressing Tubulin-GFP at near-endogenous level were immunostained with rabbit anti-GFP PABG1 antibody and Nano-Secondary 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.

Higher resolution with Nano-Secondary anti-rabbit IgG compared to conventional secondary antibodies: Left: Formation of a small, precise complex of Nanobodies (green) & primary antibody (grey). Right: Formation of a large, arbitrary complex of multiple polyclonal secondaries (green) & primary rabbit antibody.

Nano-Secondary alpaca anti-rabbit IgG VHH Alexa Fluor® 488 was applied in conjunction with rabbit anti-Lamin B1 antibodies for immunostaining of nuclear lamina (green) in HeLa cells. Scale bar, 20 µm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

Nano-Secondary alpaca anti-rabbit IgG VHH Alexa Fluor® 568 was applied together with rabbit anti-Ki67 antibodies for detection of Ki67 (red) in HeLa cells. Scale bar, 20 µm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

HeLa cells were immunostained with rabbit anti-Lamin B1 antibodies and Nano-Secondary alpaca anti-rabbit IgG 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.

ChromoTek's Nano-Secondary alpaca anti-rabbit IgG 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.

One-step immunostaining (top row) vs. sequential immunostaining (bottom row) of HeLa cells. Anti-β-Actin (left column) and anti-LaminB1 (right column) primary rabbit antibodies + Nano-Secondary alpaca anti-rabbit IgG Alexa Fluor® 647. Scale bar, 20 μm.

Multiple Nano-Secondaries can be applied for multiplex fluorescent Western blotting. This allows multiple targets to be analyzed simultaneously on the same blot at the same time. Multiplex fluorescent Western blot of GFP-TOM70, ß-Tubulin, and GFP in HEK293T cell lysate. Western blot membrane was simultaneously incubated with primary antibodies and Nano-Secondaries. Green: rabbit anti-GFP (ChromoTek PABG1) + Nano-Secondary alpaca anti-rabbit IgG Alexa Fluor® 488. Magenta: mouse anti-ß-Tubulin + Nano-Secondary alpaca anti-mouse IgG2b Alexa Fluor® 647.

Western blot analysis of EGFP (EGFP-250, ChromoTek) added to HEK293T cell lysate. Detection with rabbit anti-GFP antibody PABG1 (ChromoTek) and Nano-Secondary alpaca anti-rabbit IgG Alexa Fluor® 488.

Description
Nano-Secondary^® Alpaca anti-rabbit IgG, recombinant V_HH is an anti-rabbit IgG specific secondary antibody. It consists of of a mixture of 2 anti-rabbit Nanobodies that bind to rabbit IgG with high affinity & specificity. The product belongs to the Nano-Secondaries^® family.

Target: Rabbit
Host: Alpaca-derived, recombinantly produced in bacteria
Specificity: Fab and Fc fragments of rabbit IgG
No cross-reactivity to: Mouse, rat, sheep, goat, and guinea pig serum
Cross-reactivity to: Human and macaque serum
Formats:
Conjugates: Alexa Fluor^® 488, 568, and 647
Unconjugated: 2x Cystein

Applications
Immunofluorescence
Super-resolution microscopy
Western blotting
Flow cytometry

Fast one-step immunostaining
Simultaneous incubation of primary antibody and alpaca anti-rabbit IgG, recombinant V_HH

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

Product type
Secondary Nanobody respectively V_HH

Target/ Specificity
Fc [CTK0101] and Fab [CTK0102] fragments of rabbit IgG

Format
Alpaca single domain antibodies, monovalent

Host
Alpaca-derived, recombinantly produced in bacteria

Cross-reactivity
No cross-reactivity to mouse, rat, sheep, goat, and guinea pig IgG
Cross-reactivity to human and macaque IgG

Immunogen
Purified rabbit IgG

Clonality
Conjugated: Mixture of 2 monoclonal Nanobodies [CTK0101], [CTK0102]
Unconjugated: Monoclonal Nanobodies [CTK0101] or [CTK0102]

Clones
CTK0101, CTK0102

Conjugates
Site-directed conjugation to Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 647 or unconjugated

Recommended dilution
Immunofluorescence 1:1,000
Super-resolution microscopy 1:1,000
Western blot 1:1,000
Optimal working concentration is application-dependent and should be determined by testing a dilution range from 1:250 to 1:2,000.
Note: Image acquisition time may have to be optimized.

Synonyms
Alpaca single domain antibody, V_HH, Nanobody, binding domain of single domain antibody, Nano antibody

Validation
Application validated for immunofluorescence, SRM, and Western blotting
Determination of cross-reactivity, sequence, affinity, melting point, and degree of labeling (DOL)

Affinity (K_D)
CTK0101: K_D = 0.2 nM, CTK0102: K_D = 1.2 nM

Degree of labeling (DOL)
2 fluorophores per Nanobody

Purity
Recombinantly expressed and purified

Form
Buffered aqueous solution

Storage buffer
10 mM HEPES pH 7.0, 500 mM NaCl, 5 mM EDTA, Preservative: 0.09 % Sodium azide

Concentration
0.5 g/ L

Size
10 µL; 100 µL

How to cite these products
RRIDs:
Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 488 [CTK0101, CTK0102]: AB_2827585
Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 568 [CTK0101, CTK0102]: AB_2827586
Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 647 [CTK0101, CTK0102]: AB_2827587
Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, for 2x Cys conjugation [CTK0101]: AB_2864261
Nano-Secondary® alpaca anti-rabbit IgG, recombinant VHH, for 2x Cys conjugation [CTK0102]: AB_2864262

Storage instructions
Shipped at ambient temperature. Store at -20°C short term or -80°C long term. Aliquot upon delivery. Avoid freeze-thaw cycles. Stable for 6 months after arrival.

Protocol
Protocol Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 488 [CTK0101, CTK0102] (PDF)

Protocol Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 568 [CTK0101, CTK0102] (PDF)

Protocol Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 647 [CTK0101, CTK0102] (PDF)

Protocol Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, for 2x Cys conjugation [CTK0101] (PDF)

Protocol Nano-Secondary® alpaca anti-rabbit IgG, recombinant VHH, for 2x Cys conjugation [CTK0102] (PDF)

Brochure
Nano-Secondaries product brochure (PDF)

Application note
One-step incubation with Nano-Secondaries (PDF)

Blocking recommendation for immunostaining with Nano-Secondaries (PDF)

Maleimide chemistry labeling protocol of Nanobodies (PDF)

SDS
SDS Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 488 [CTK0101, CTK0102] (PDF)

SDS Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 568 [CTK0101, CTK0102] (PDF)

SDS Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 647 [CTK0101, CTK0102] (PDF)

SDS Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, for 2x Cys conjugation [CTK0101] (PDF)

SDS Nano-Secondary® alpaca anti-rabbit IgG, recombinant VHH, for 2x Cys conjugation [CTK0102] (PDF)

Benefits

Reduced incubation and hands-on time because the Alpaca anti-rabbit IgG, recombinant V_HH can be incubated together with the rabbit primary antibody
Higher resolution imaging due to the Alpaca anti-rabbit IgG, recombinant V_HHs’ 10 times smaller size than conventional secondary antibodies. The small size enables better tissue penetration, antigen access and decreases the distance between the epitope and label.
Cleaner Images: The Alpaca anti-rabbit IgG, recombinant V_HH has a high specificity and low background:
We select only V_HHs with the desired specificity during development; V_HHs that cross-react to other commonly used species’ IgGs are eliminated. Hence, ChromoTek's Nano-Secondaries have an extreme low background; any kind of pre-adsorption is not needed.
Multiplexing: the Alpaca anti-rabbit IgG, recombinant V_HH does not cross-react to other frequently used IgGs and makes multiplexing possible.

One-step immunostaining protocol saves time

ChromoTek’s Alpaca anti-rabbit IgG, recombinant V_HH consists of monovalent Nanobodies with very high affinity. The monovalent Alpaca anti-rabbit IgG, recombinant V_HH enables the simultaneous incubation together with rabbit primary antibodies.
This one-step immunostaining saves washing steps and incubation time. 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 Alpaca anti-rabbit IgG, recombinant V_HH, too.

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 ChromoTek’s Alpaca anti-rabbit IgG Nano-Secondary has been carefully tested. It is co-reactive to human IgG. It shows no cross-reactivity to mouse, rat, sheep, goat, and guinea pig serum. Cross-reactivity to macaque serum has been determined. Because of this high specificity our Nano-Secondaries do not require any kind of pre-adsorption.

Precise and reproducible staining

Site specific binding of 4 Alpaca anti-rabbit IgG, recombinant V_HHs per rabbit primary IgG antibody
Stoichiometric labelling of 2 Alexa Fluor dyes per V_HH respective Nanobody
Fully characterized and validated
Recombinantly produced, virtually without lot-to-lot variation
Manufacturing process is totally animal free, though V_HHs respective Nanobodies are generated from immunization of alpacas and llamas.

Site-directed labeling of Nano-Secondaries

ChromoTek's Nano-Secondaries are conjugated to AlexaFluor 488, 568, or 647. For custom dyes, we offer unconjugated Nano-Secondaries. They have 2 Cystein residues for conjugation with thiol-reactive dyes, e.g. maleimide. Please note that ChromoTek's Nano-Secondaries are often a mix of two different monoclonal Nanobodies whereas the unconjugated Nano-Secondaries are always sold as monoclonals and not pre-mixed.

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