ChromoBlog

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).

Fluorescent proteins (FPs) like GFP or RFP and their derivatives are commonly used for virus research. Frequently, they are applied as fluorescent markers in microscopy or cell sorting experiments and as protein tags for protein purification, immunoprecipitation or protein-protein interaction assays. Here, we provide a short outline that describes how FPs have been used in the current SARS-CoV-2 research. Please note that this summary does not provide a complete overview; also, many referenced papers are pre-prints without peer reviews.

SNAP-tag and CLIP-tag are self-labeling protein tags. The SNAP-tag protein and the CLIP-tag protein can be fused to a protein of interest (POI) and used for cellular and biochemical analysis.

HaloTag is a protein tag. The HaloTag protein can be fused to your protein of interest (POI) which enables it for cellular and biochemical analysis. It can also be used for live cell imaging.

Why Spot-Tag can be used to purify any protein

 

Introduction

Proteins often have very different individual characteristics and requirements for manipulation. Not every purification tag in combination with the desired purification resin is suitable for every protein. Some proteins are sensitive towards wash or elution buffers, while others are difficult to purify because the selected affinity resin can be contaminated by host cell proteins. Taking these points into consideration, the selection of both the affinity tag and the affinity resin can have a strong impact on protein purity and yield, which is often dependent on the target protein. Requirements for an affinity resin comprise (i) high purity (minimal contaminations), (ii) high yield of the purified protein, and (iii) a broad buffer stability. Ideally, affinity tags should be (iv) inert and (v) short in order to not or minimally affect the purified protein.

What’s a Chromobody?

Chromobodies® are fluorescent nanoprobes for real-time and live-cell imaging of intracellular endogenous proteins. A Chromobody is a small intracellular functional antibody. It consists of a V_HH/Nanobody that is genetically fused to a fluorescent protein. Plasmids encoding for Chromobodies can be transiently transfected into cells and the Chromobody protein is intracellularly expressed.

Our Sales Rep Stephanie Dennison answers one of the most frequent customer questions:

Nano-Traps are a great tool for easy, clean immunoprecipitation, but certain VHHs bind so effectively that it can be difficult or impossible to do native elution without a TEV cleavage site. After receiving feedback from our customers, we understood the need for capturing proteins in applications like affinity purification and then getting them off the bead while preserving their structure and function. We launched Nano-Caps in early 2020 starting with Spot-Cap^®.