ChromoBlog

What GFP-Trap should I use for my immunoprecipitation?

Posted by Dr. Astrid Sitte on Jan 20, 2020 4:42:38 PM

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:

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Topics: GFP-Trap, GFP Immunoprecipitation, GFP Nanobody, Dynabeads

GFP-Trap Dynabeads: Most cited GFP-Nanobody conjugated to Dynabeads

Posted by Christoph Eckert on Jan 17, 2020 11:27:30 AM

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.

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Topics: GFP Immunoprecipitation, GFP Nanobody, Dynabeads

How to overcome the limitations of antibody-based affinity resins for protein purification

Posted by Dr. Astrid Sitte on Dec 18, 2019 2:19:42 PM

Introduction

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Topics: affinity resin, affinity purification, Spot-Tag, Spot Nanobody, protein purification, epitope tag

Tags for affinity purification

Posted by Christoph Eckert on Nov 18, 2019 2:56:52 PM

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.

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Split fluorescent protein technology

Posted by Klaus Herick on Nov 12, 2019 2:57:15 PM

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).

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Bispecific T cell engagers

Posted by Christoph Eckert on Sep 13, 2019 1:04:52 PM
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:

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Topics: VHH, Nanobody, Bispecific, T cell

Spot Capture and Detection Peptide Tag and Nanobody

Posted by Klaus Herick on Sep 9, 2019 1:37:29 PM

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-VHH. 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.

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Topics: Spot-Tag, Spot Nanobody, Spot-Label

Which are 2018's most used Antibodies?

Posted by Klaus Herick on Aug 26, 2019 4:29:03 PM

 
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Topics: GFP-Trap, GFP Immunoprecipitation, GFP VHH, GFP Nanobody

iCLIP for the thorough analysis of mRNA:protein interactions

Posted by Christoph Eckert on Aug 14, 2019 11:41:21 AM

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.

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Topics: GFP, iCLIP

What is one-step immunostaining?

Posted by Dr. Astrid Sitte on Jul 26, 2019 2:45:28 PM

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.

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Topics: live cell imaging, Secondary antibody, Nano-Secondaries, One-step immunostaining, staining

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