ChromoBlog

More and more publications are coming up using ChromoTek’s Spot-Tag system, the first peptide-tag specific Nanobody system for universal capture & detection applications.

Interaction of Spot-Nanobody (green) with Spot Peptide

Myc-tag is a peptide tag derived from the c-Myc protein. The Myc-tag can be used for many capture and detection applications such as immunoprecipitation, immunofluorescence and protein purification.

Nanobody-Fc fusion - An alternative Nanobody format that combines its special epitope binding properties with the detection options for Fc-domains

What are heavy chain antibodies?

When time in the lab is limited, learn how to save precious hours on your immunoprecipitation, immunofluorescence and western blotting experiments.

Due to the current situation with the COVID-19 pandemic, many researchers around the world are faced with restrictions on time and resources in the lab. As we adapt to these changes, the pressure is on to find ways to save time without compromising on results. Proteintech and ChromoTek offer solutions to save you hours on your current immunoassay protocols, helping you achieve publication worthy results in a fast and reliable manner.

The conjugation of the binding domains of camelid heavy chain antibodies, also known as V_HHs or Nanobodies, to fluorescent dyes is generating versatile tools for fluorescence microscopy applications like immunofluorescence (IF) and super resolution microscopy (SRM): The small size Nanobodies provide better penetration into tissue and dense cell compartments, plus the small label to epitope displacement provides higher resolution. In addition, the Nanobodies’ high affinity binding to epitope makes them attractive probes for other assays like ELISA.

ChromoTek, part of Proteintech group, provides multiple fluorescent dye (Alexa Fluor^® and ATTO) conjugated Nanobodies that are optimized by application:

Using both Proteintech and Chromotek antibodies, Davy and colleagues published a landmark paper in Science late in 2020, detailing how Neuropilin-1 is required for SARS-CoV-2 host cell entry.

V5 tag is a short peptide tag for detection and purification of proteins. The V5 tag can be fused/cloned to a recombinant protein and detected in ELISA, flow cytometry, immunoprecipitation, immunofluorescence, and Western blotting with antibodies and Nanobodies.

Parts of them are used as epitope tags

Epitope tags, i.e. short peptide tags are commonly used, versatile, and convenient tools for multiple capture and detection applications including immunofluorescence, immunoprecipitation, affinity purification, and Western blotting.

ChromoTek's Nano-Traps are optimized for the immunoprecipitation (IP) of proteins and their interacting factors. Nano-Traps comprise of a Nanobody/ V_HH conjugated to beads. For 3 Nano-Traps, e.g. GFP-Trap^®,  RFP-Trap^®, and Spot-Trap^®, we offer different types of beads:

• Agarose beads
• Magnetic Agarose beads
• Dynabeads™

Immunoprecipitation with GFP-Trap.
I: Input, FT: Flow-Through, B: Bound

Based on the properties of the different beads (see table), we recommend:

• Agarose beads for very low background and high binding capacity IP
• Magnetic Agarose beads for magnetic separation and high binding capacity IP
• Dynabeads for IP of very large proteins/complexes
  

Characterization of antibodies

Essential aspects of how antibodies (Abs) bind to their targets are their affinity and binding kinetics. In order to determine these parameters, different biophysical methods such as Surface Plasmon Resonance (SPR), Bio-Layer Interferometry (BLI), MicroScale Thermophoresis (MST), or Enzyme-linked Immunosorbent Assay (ELISA) can be applied. Some of these methods require the immobilization of the antibody on a surface and the titration of the target antigen to measure the dissociation constant K_D and the association and dissociation rates k_a and k_d (also called on- and off rates k_on and k_off).