ChromoBlog

GFP-Booster and RFP-Booster conjugated to Alexa Fluor® 488, 568 and 647:

• Stronger signal in confocal and standard fluorescence microscopy
• More options in super-resolution microscopy including STORM and MINFLUX
• Constant degree of labeling (DOL = 2 dyes per Booster) for higher resolution
• More than 95% labeling efficiency

Introduction

Blocking is an essential step during the preparation of a sample for immunofluorescence detection. Blocking improves the sensitivity by reducing nonspecific background and therefore increases image quality. Insufficient blocking results in higher background noise and over-blocking can even mask the specific signal.

ChromoTek GFP-Trap^® is optimized for the immunoprecipitation of GFP-tagged proteins and their interacting factors.

The GFP-Trap consists of the ChromoTek anti-GFP Nanobody/ VHH that is coupled to 3 different types of beads:

• Agarose beads
• Magnetic Agarose beads
• Dynabeads™

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

GFP-Trap^® Dynabeads is optimized for the immunoprecipitation (IP) of large GFP-tagged proteins and for Co-IP of protein complexes. It consists of ChromoTek’s established anti-GFP Nanobody conjugated to Dynabeads™. Hence, also GFP-Trap Dynabeads has the very high affinity of 1 pM like GFP-Trap Agarose and Magnetic Agarose.

Introduction

Affinity tags are very useful tools for protein purification. Fused to the protein of interest, they streamline the purification process by binding to a tag-specific resin. Obviously, tag selection is an important step as the purification tag can affect expression level, solubility, facilitate correct folding, protect from proteolysis, and re-direct proteins to a cellular compartment. In addition, the purification tag determines the affinity resin used.

ChromoTek scientists Michael Metterlein and Christian Linke-Winnebeck have published a whitepaper that provides a comprehensive overview with key developments in the field of split fluorescent protein technology. It also includes a selection of case studies on how ChromoTek’s Nano-Traps have been applied to exploit the full potential of this technology for example in protein-protein interaction studies. Particularly, the ChromoTek GFP-Trap has been successfully applied to multiple assays using different split GFP variants. Assay types include protein self-complementation, bimolecular fluorescence complementation (BiFC), tripartite fluorescence complementation (TriFC), and bimolecular complementation affinity purification (BiCAP).

Bispecific T cell engagers are bispecific antibodies designed to link T cells with cancer cells. The simultaneous binding of the bispecific antibody to T cell and tumor cell initiates the activation of the T cell. For this purpose, the bispecific antibody carries two antigen binding domains against the T cell (often CD3 T cell receptor) and against a cell surface protein of the tumor cell.

ChromoTek offers two bispecific T cell engagers to beta-testers:

The Spot-Nanobody (green) binds to the Spot-Tag sequence motif PDRVRAVSHWSS. Upon binding, the Spot-Tag peptide is embedded on the surface of the Spot-Nanobody and becomes a β-sheet extension of the Spot-V_HH. Defined interactions of the Spot-Nanobody’s side chains to the Spot-peptide determine specificity. In addition, the Spot-peptide is clamped by two amino acid side chains of the Spot-Nanobody. This binding mechanism elucidates why the Spot-Nanobody binds with high affinity to the Spot-Tag.