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
- Benefits for pulldown and purification
- Advantages for microscopy
Camelids such as dromedaries, llamas, and alpacas possess an immune repertoire of three subclass IgG antibodies: IgG1, IgG2, and IgG3. IgG1 is a conventional IgG composed of two heavy chains and two light chains. IgG2 and IgG3 are heavy chain only IgGs antibodies (HCAbs) that can be distinguished by their hinge regions. These HCAbs lack the CH1 domain of the heavy chain and are devoid of any light chain. 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.
Camelids comprise so-called old-world camelids, i.e. Camelus bactrianus and Camelus dromedarius and new-world camelids, i.e. Lama pacos, Lama glama, and Lama vicugna.
- 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
- 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
- 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
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 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).
- 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).
- Advanced coupling to matrix to reduce non-specific binding
- More than 1,600 scientific publications
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:
- 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
- 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
- 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