Alpaca anti-mouse IgG1, recombinant VHH

Anti-mouse IgG1 Nano-Secondary: Well-defined and characterized immunostaining. Primary anti-mouse IgG1 antibody (grey) with 2X2 monoclonal mouse Fc- specific Nanobodies (green) bound. In total, 8 fluorophores (red stars) label the mouse IgG1 primary antibody.

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.

Western blot analysis of EGFP (EGFP-250, ChromoTek) added to HEK293T cell lysate. Detection with anti-GFP mouse IgG 1 antibody and alpaca anti-mouse IgG1 VHH Alexa Fluor® 647.

One-step staining (left) vs. sequential staining (right) of HeLa cells with anti-COX4 (mitochondria) mouse IgG1 monoclonal primary antibody + alpaca anti-mouse IgG1 VHH Alexa Fluor® 647 (magenta). Cell nuclei are stained with DAPI (blue). Scale bar, 20 µm.

The anti-mouse IgG1 Nano-Secondary is subclass-specific and does not cross-react with IgGs from other commonly used species (here rabbit) and with mouse IgG2b and IgG3 subclasses.

HeLa cells were immunostained with anti-Vimentin mouse IgG1 antibody and alpaca anti-mouse IgG1 VHH Alexa Fluor® 647 (grey). Nuclei were stained with DAPI, blue. Scale bar, 10 µm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.

One-step immunostaining is the simultaneous incubation of mouse IgG1 primary antibody and anti-mouse IgG1 Nano-Secondary. This method reduces incubation and hands-on time. Simultaneous incubation also supports multiplexing, tissue penetration, and cell staining for flow cytometry.

Flow cytometry with anti-mouse Nano-Secondaries. Jurkat CD3+ and Jurkat CD3- cell lines were mixed and immunostained live with anti-CD3 mouse IgG1 and alpaca anti-mouse IgG1 VHH Alexa Fluor® 647 (1:600). Two cell populations can be clearly distinguished with a 2-log shift: CD3-positive cells (in green) and CD3-negative cells (in grey).

Immunostaining with Alpaca anti-mouse IgG1 Alexa Fluor 647 Nano-Secondaries for FACS. 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.

Description
Alpaca anti-mouse IgG1, Fc-specific recombinant V_HH is an anti-mouse IgG subclass specific antibody. This secondary antibody product consists of Nanobodies that bind to mouse IgG1 with high affinity & specificity. The product belongs to the Nano-Secondaries^TM family.

Target: Mouse IgG1
Host: Alpaca-derived, recombinantly produced in bacteria
Specificity: Fc fragment of mouse IgG1
No cross-reactivity to: Goat, guinea pig, rabbit, rat, sheep, human, and macaque serum and to mouse IgG2a, IgG2b, IgG2c, and IgG3
Clonality: Biclonal, mixture of 2 monoclonal Nanobodies
Conjugates: Alexa Fluor^® 488, 568 and 647

Applications
Immunofluorescence
Super-resolution microscopy
Western blotting
Flow cytometry

Fast one-step immunostaining
Simultaneous incubation of mouse IgG1 primary antibody and Alpaca anti-mouse IgG1, recombinant V_HH

Product	Size	Code	Price	Buy
Product Alpaca anti-mouse IgG1, Fc-specific recombinant VHH, Alexa Fluor® 488	Size 10 µL	Code sms1AF488-1-10	Price $ 100	Buy +
Product Alpaca anti-mouse IgG1, Fc-specific recombinant VHH, Alexa Fluor® 488	Size 100 µL	Code sms1AF488-1-100	Price $ 365	Buy +
Product Alpaca anti-mouse IgG1, Fc-specific recombinant VHH, Alexa Fluor® 568	Size 10 µL	Code sms1AF568-1-10	Price $ 100	Buy +
Product Alpaca anti-mouse IgG1, Fc-specific recombinant VHH, Alexa Fluor® 568	Size 100 µL	Code sms1AF568-1-100	Price $ 365	Buy +
Product Alpaca anti-mouse IgG1, Fc-specific recombinant VHH, Alexa Fluor® 647	Size 10 µL	Code sms1AF647-1-10	Price $ 100	Buy +
Product Alpaca anti-mouse IgG1, Fc-specific recombinant VHH, Alexa Fluor® 647	Size 100 µL	Code sms1AF647-1-100	Price $ 365	Buy +

Product type
Secondary Nanobody (secondary V_HH)

Target/ Specificity
Mouse IgG1; this Nanobody mixture recognizes Fc fragments of mouse IgG1

Format
Alpaca single domain antibodies, monovalent

Host
Alpaca-derived, recombinantly produced in bacteria

Cross-reactivity
No cross-reactivity to rabbit, rat, goat, sheep, guinea pig, human, and macaque serum and to mouse IgG2a, IgG2b, IgG2c, and IgG3

Immunogen
Purified mouse IgG1

Clonality
Biclonal: mixture of 2 monoclonal Nanobodies

Clones
V_HH302, V_HH305

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

Recommended dilution
Immunofluorescence 1:500
Super-resolution microscopy 1:500
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,subclass specificity, sequence, affinity, melting point, and degree of labeling (DOL)

Affinity (K_D)
V_HH302: K_D =0.13 nM, V_HH305: K_D = 0.63nM

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

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 Alpaca anti-mouse IgG1, Fc-specific recombinant VHH, Alexa Fluor® 488 (PDF)
Protocol Alpaca anti-mouse IgG1, Fc-specific recombinant VHH, Alexa Fluor® 568 (PDF)
Protocol Alpaca anti-mouse IgG1, Fc-specific recombinant VHH, Alexa Fluor® 647 (PDF)

Brochure
Nano-Secondaries product brochure (PDF)

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

SDS
SDS Alpaca anti-mouse IgG1 Alexa Fluor 488 (PDF)
SDS Alpaca anti-mouse IgG1 Alexa Fluor 568 (PDF)
SDS Alpaca anti-mouse IgG1 Alexa Fluor 647 (PDF)

Benefits

Faster staining
One-step staining saves time due to simultaneous incubation of mouse IgG1 primary antibody and Alpaca anti-mouse IgG1, recombinant V_HH.

Higher resolution
Alpaca anti-mouse IgG1, recombinant V_HH is about 10 times smaller than conventional secondary antibodies. The small size enables better tissue penetration, antigen access, and decreases the distance between epitope and label.

Cleaner Images
Nano-Secondaries have a high specificity and low background:

We select only V_HHs with the desired specificity during development; V_HHs that cross-react to IgGs from other commonly used species and to other IgG subclasses are eliminated. Therefore, our Nano-Secondaries have a very low background and hence do not require any kind of pre-adsorption.

Multiplexing
Alpaca anti-mouse IgG1, recombinant V_HH is subclass-specific and does not cross-react to IgGs from other commonly used species or other mouse IgG subclasses. This high specificity in combination with low background enables multiplexing.

Time savings by one-step immunostaining

ChromoTek’s Alpaca anti-mouse IgG1, recombinant V_HH consists of 2 monovalent Nanobodies with very high affinity. Hence, they enable one-step immunostaining: the mouse IgG1 primary antibody and alpaca anti-mouse IgG1, recombinant V_HH can be incubated together.

This method saves incubation time and washing steps.

Of course, Nano-Secondaries can be used according to standard immunostaining protocols following a sequential incubation of mouse IgG1 primary antibody and alpaca anti-mouse IgG1, recombinant V_HH, too.

Simultaneous incubation also supports:

multiplexing
tissue penetration
cell staining for flow cytometry

Better imaging

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

No cross-reactivity to IgG from other commonly used species and mouse IgG subclasses

The cross-reactivity of ChromoTek’s Alpaca anti-mouse IgG1 Nano-Secondary has been carefully tested. It shows no cross-reactivity to the mouse subclasses IgG2a, IgG2b, IgG2c, and IgG3, and no cross-reactivity to rabbit, rat, goat, sheep, guinea pig, human, and macaque serum.

Precise and reproducible staining

Site specific binding of 4 Nanobodies per mouse IgG1 primary antibody
Stoichiometric labelling of 2 Alexa Fluor dyes per Nanobody
Characterized and validated: DNA and protein sequences, affinity, melting temperature
Recombinantly produced, virtually without lot-to-lot variation
Manufacturing process is totally animal free, though Nanobodies are generated from immunization of alpacas and llamas.

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