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Nano-Secondary alpaca anti-mouse IgG1

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Nano-Secondary anti-mouse IgG1: 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 + Nano-Secondary alpaca anti-mouse IgG1 VHH Alexa Fluor® 647. Green: Mouse IgG2b anti-Lamin + Nano-Secondary alpaca anti-mouse IgG2b VHH Alexa Fluor® 488. Red: Mouse IgG3 anti-MOT + Nano-Secondary 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 Nano-Secondary alpaca anti-mouse IgG1 Alexa Fluor® 647.
One-step staining (left) vs. sequential staining (right) of HeLa cells with anti-COX4 (mitochondria) mouse IgG1 monoclonal primary antibody + Nano-Secondary alpaca anti-mouse IgG1 Alexa Fluor® 647 (magenta). Cell nuclei are stained with DAPI (blue). Scale bar, 20 µm.
The Nano-Secondary 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.
Vimentin in HeLa cells: The cells were stained with monoclonal anti-Vimentin mouse IgG1 antibody and Nano-Secondary alpaca anti-mouse IgG1 Alexa Fluor® 488 and recorded with Leica GSDIM system. The image is a courtesy of Dr. Leila Nahidiazar, Dr. Jop Kind, and Prof. Kees Jalink.
Multiplexed immunostaining of HeLa cells with 3 subclass-specific alpaca anti-mouse Nano-Secondaries. Grey: Mouse IgG1 anti-Vimentin + Nano-Secondary alpaca anti-mouse IgG1 Alexa Fluor® 647. Green: Mouse IgG2b anti-Lamin + Nano-Secondary alpaca anti-mouse IgG2b Alexa Fluor® 488. Red: Mouse IgG3 anti-MOT + Nano-Secondary alpaca anti-mouse IgG3 Alexa Fluor® 568. Scale bar, 10 µm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
HeLa cells were immunostained with anti-Vimentin mouse IgG1 antibody and Nano-Secondary alpaca anti-mouse IgG1 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 Nano-Secondary anti-mouse IgG1. 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 Nano-Secondary alpaca anti-mouse IgG1 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 + Nano-Secondary alpaca anti-mouse IgG1 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 VHH 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® 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
Formats:
Conconjugated: Alexa Fluor® 488, 568 and 647
Unconjugated: 2x Cystein for maleimide conjugation

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 VHH

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

Product type
Secondary Nanobody (secondary VHH)

Target/ Specificity
Fc fragments of mouse IgG1 antibodies

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
Conjugated: Mixture of 2 monoclonal Nanobodies [CTK0103], [CTK0104]
Unconjugated: Monoclonal Nanobodies [CTK0103] or [CTK0104] for maleimide conjugation

Clones
CTK0103, CTK0104

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

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, VHH, 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 (KD)
CTK0103: KD =0.13 nM, CTK0104: KD = 0.63nM

Degree of labeling (DOL)
2 fluorophores per Nanobody

Purity
Recombinantly expressed and purified

Form
Buffered aqueous solution

Storage buffer

Conjugated: 10 mM HEPES pH 7.0, 500 mM NaCl, 5 mM EDTA 0.09 % sodium azide

Unconjugated: 10 mM HEPES pH 7.0, 500 mM NaCl, 1 mM TCEP, 0.09 % sodium azide

Concentration
0.5 g/ L

Size
10 µL; 100 µL

How to cite these products
RRIDs:
Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 488 [CTK0103, CTK0104]: AB_2827578

Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 568 [CTK0103, CTK0104]: AB_2827579

Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 647 [CTK0103, CTK0104]: AB_2827580

Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, for 2x Cys conjugation [CTK0103]: AB_2864263

Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, for 2x Cys conjugation [CTK0104]: AB_2864264

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.

Benefits

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

Higher resolution
Alpaca anti-mouse IgG1, recombinant VHH 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 VHHs with the desired specificity during development; VHHs 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 VHH 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 VHH 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 VHH 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 VHH, 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.

Site-directed labeling of Nano-Secondaries

ChromoTek's Nano-Secondaries are conjugated to AlexaFluor 488, 568, or 647. For alternative dye conjugation, 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.

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