The ChromoTek Alpaca Antibody Advantage

Here you find the general and ChromoTek-specific advantages and benefits of alpaca Nanobodies for proteomics and cell biology. Three chapters are discussed:

 

General advantages of alpaca antibodies

Next to conventional IgG antibodies, which are composed of two heavy chains and two light chains, the immune repertoire of alpacas contains so-called heavy chain only IgGs. These antibodies lack the CH1 domain of the heavy chain and are devoid of any light chain1. The antigen binding domain of a heavy chain only IgG is called VHH or Nanobody. VHHs have several general advantages; ChromoTek’s Nanobodies and Nanobody-based research tools, however, have even more advantages: their high level of performance, characterization, and validation.

Small-size

  • Our Nanobodies are 15 kDa in size and about 10-fold smaller than conventional IgG antibodies (150 kDa)
  • Higher labelling density
  • Higher image resolution because of very small 2 nm epitope label displacement

Epitope-access

  • Good epitope access even in crowed cellular environments
  • Considerable better penetration into tissues, organs, and animals

Robust binding molecules:

  • Chemical and thermal stability
  • Long shelf life time
  • Room temperature shipment

Reliable & reproducible VHHs:

  • Recombinant production
  • Monoclonal
  • Robust conjugation
  • Quality controlled

Validated affinity reagents:

  • Sequence and structure validated
  • Function validated
  • Published in more than 1,600 scientific publications
  • Virtual no production batch to batch variation

Benefits for pulldown and purification

For immunoprecipitation, affinity purification, and additional biochemical applications, the ChromoTek Nanobodies and Nano-Traps (= Nanobody coupled to agarose bead) do provide multiple advantages for superior performance:

Single band purification

  • No contamination of IgG antibody heavy and light chains
  • Highly specific binding
  • Low background

High stability

  • High chemical stability allows for very harsh buffer and washing conditions, e.g. up to 8 M urea. ChromoTek’s Nanobodies and Nano-Traps are individually tested for binding in detergents, salt, reducing agents, and chaotropes (see specifications of individual Nano-Trap).
  • High thermal stability allows binding at elevated temperatures of up to 83 °C (see specifications of individual Nano-Trap).

High affinity

  • The kinetic properties of our Nanobodies have been thoroughly determined. Very high affinities with dissociation constants (KD) in a range from 1 pM to 7 nM have been determined. These high affinities enable the effective binding of proteins even at low concentration, i.e. at endogenous protein expression level (see specifications of individual Nano-Trap).

Ready to use

  • ChromoTek’s VHHs are covalently bound to agarose and magnetic agarose beads
  • Nano-Traps are ready for pulldown or affinity purification
  • High binding capacity (see specification of individual Nano-Trap).

Low background

  • Advanced coupling to matrix to reduce non-specific binding

Proven

  • More than 1,600 scientific publications

Advantages for Microscopy

ChromoTek’s Nanobodies that are coupled to fluorescent dyes are called Nano-Boosters or Nano-Labels. These provide multiple advantages for superior imaging performance in multiple immunofluorescence applications such as epifluorescence, confocal, and super resolution microscopy:

Small-size

  • Our Nanobodies are about 10-fold smaller than conventional IgG antibodies and 2 nm in size
  • 100 µg of Nanobody/ Nano-Booster corresponds to 1,000 µg of conventional IgG antibody

Higher labelling density

  • More molecules of Nano-Boosters/ Nano-Labels than by bulky IgG molecules can access the antigens in crowded cellular environments.

Higher image resolution

  • Higher image resolution because of very small 2 nm epitope label displacement of a monovalent Nano-Booster/ Nano-Label, which is at least 10 times less than (a complex) of conventional antibodies/ secondaries
  • Ideal for super resolution microscopy applications

Epitope-access

  • Considerable better penetration into tissues, organs, and animals:
  • ChromoTek Nano-Boosters GFP-Booster and RFP-Booster have been used to label whole genetic modified mice after clearing procedures

No clustering

  • Monovalent Nanobodies do not cluster unlike conventional IgG antibodies, which are bivalent and thus may form large clusters

Avidity effects from bivalent formats

  • Bivalent Nanobody formats do gain considerably from avidity effects and provide better labelling performance when needed