ChromoBlog

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.

Immunostaining in HeLa cells low expressing Tubulin-GFP.
Left: GFP signal of Tubulin-GFP (green) and DAPI stain (blue);
Right: Tubulin-GFP detection by GFP-Booster coupled to Alexa Fluor 647

We currently offer our GFP- and RFP-Booster conjugated to two different far-red dyes:

Alexa Fluor® 647 and  ATTO647N.

Nanobodies are the binding domains of heavy chain only antibodies from Camelids. Nanobodies can be recombinantly produced in bacterial and other animal-free expression systems depending on the actual Nanobody construct. In contrast, classical IgG antibodies are composed of two heavy chains and two light chains and are traditionally produced using hybridoma technlogies or are isolated from a host’s blood.