ChromoBlog

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

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

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.

 

Crystal structure of the anti-GFP VHH-Green Fluorescent Protein complex.
The GFP Nanobody is displayed blue and the GFP in green color.

UV crosslinking techniques are the method of choice for a comprehensive analysis of in-vivo-mRNA targets of an RNA-binding protein (RBP). In the recent publication of Olgeiser et al. (2019), the authors applied individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) to study fungal mRNA transport. For this approach, they have used strains expressing GFP-tagged versions of the two RBPs Grp1 and Rrm4; an optimized protocol was developed to uncover that Grp1 and Rrm4 conjointly bind thousands of shared target messenger ribonucleoproteins (mRNPs) in the fungus U. maydis. The protein:RNA complexes were immunoprecipitated in a multiple detergent containing buffer using ChromoTek’s GFP-Trap Magnetic Agarose. This is a transcriptome‐wide view to an endosomal mRNA transport machinery.

Simultaneous immunostaining, also called one-step immunostaining vs. sequential immunostaining. Nano-Secondaries stain different primary antibodies equally well in one-step staining and sequential staining.

HeLa cells were immunostained with different primary antibodies and Nano-Secondaries Alexa Fluor® 647 (1:1,000, magenta). Cell nuclei were stained with DAPI (blue). Scale bar, 20 μm.