ChromoBlog

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.

Jellyfish Green Fluorescent Protein (GFP) and its derivatives are still the most frequently used fluorescent proteins in biomedical research. Recently, additional green fluorescent proteins have been discovered in higher animals such as crustaceans and lancelets. These FPs share a common fold, but diverge widely in their primary sequence. Thus, they require novel, dedicated antibody research tools. Here is an overview about EGFP (the most commonly used GFP derivative), TurboGFP and mNeonGreen.

Chromotek’s GFP- binding protein, an anti- GFP Nanobody, is a very small and effective tool for binding and visualizing GFP-tagged proteins. In their recent publication, Modi et al. successfully functionalized quantum dots with the GFP- Nanobody (QD GFP- Nanobody). Thus, they created a small GFP- specific label with a very strong fluorescent signal.

TurboGFP is a bright green fluorescent protein used to study protein function, localization and dynamics in cells. Nanobody based research tools allow reproducible biochemical analysis including mass spectrometry and enzyme activity measurements of TurboGFP fusion proteins.

What makes on-bead digestion favorable?

Just pull down your protein of interest with immobilized nanobodies, also termed V_HHs or single domain antibodies. Then follow the on-bead digestion protocol (see below) and submit the digest to your core facility for effective mass spectrometer analysis of (co-) precipitated proteins.

Life science laboratories apply green fluorescent proteins (GFP) to study protein localization, interaction and dynamics in fluorescence microscopy. Immunoprecipitation (IP), mass spectrometry (MS), co-immunoprecipitation (Co-IP) and/or affinity purification investigate more aspects including posttranslational modifications (PTMs), DNA binding, and protein-protein interaction. Here, we compare two different antibody systems for immunoprecipitation of GFP-fusion proteins: GFP-Trap and anti-GFP IgG antibody

The ChromoTek Histone-Chromobody®* is a novel probe to visualize endogenous histone H2A-H2B heterodimer in live cells:

Recently a new bright monomeric yellow-green fluorescent protein has been published, which is called mNeonGreen. This protein has already been frequently used for mainly microscopic applications in both wide-field microscopy and super resolution microscopy. What is mNeonGreen all about?

ChromoTek is proud to introduce new tools for mNeonGreen fusion proteins: 

• anti-mNeonGreen mouse monoclonal antibody 32F6 for immunofluorescence (IF) and Western blot (WB) 
• mNeonGreen-Trap for immunoprecipitation (IP) of mNeonGreen fusion proteins. The mNeonGreen-Trap is based on an anti-mNeonGreen VHH (mNeonGreen binding protein)

Click here and request a free sample:

ChromoTek Myc-Trap® is an effectiveness optimized tool for immunoprecipitation (IP/Co-IP) and affinity purification of Myc-tagged proteins. Depending on whether you use a single Myc-tag, i.e. 1xMyc (EQKLISEEDL) or a double Myc-tag fused to the protein of interest, i.e. 2xMyc (EQKLISEEDLEQKLISEEDL) you can adjust your experiment. Here, we introduce the epitope of Myc-Trap to discuss its performance for afore mentioned applications.