ChromoBlog

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.

Introduction

Fluorescent proteins (FPs) have been used as protein tags since the mid-1990s mainly for cell biology and fluorescence microscopy. These tags have not only revolutionized cell biology by enabling the imaging of almost any protein, they are also used in biochemical applications. An important example is the immunoprecipitation and affinity purification of FP-tagged proteins, which was enabled by the development of affinity resins with high yield, purity, and affinity such as ChromoTek’s Nano-Traps (https://www.chromotek.com/products/detail/product-detail/nano-traps/).

In this blog we provide a review of

• green fluorescent proteins
• red fluorescent proteins
• self-labelling proteins, that require the covalent coupling of a fluorescent molecule

Nano-Secondaries are monoclonal Nanobodies that bind to primary antibodies in a species and subtype specific manner, i.e. Nano-Secondaries are very precise secondary antibodies. They are recombinantly produced in bacterial or other animal-free expression systems; in contrast, classical mono- or polyclonal secondary antibodies are traditionally produced using hybridoma technologies or are isolated from a host’s blood.