ChromoBlog

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.

Read More

Topics: GFP, iCLIP

Fluorescent protein tags

Posted by Christian Linke-Winnebeck on May 28, 2019 5:02:54 PM

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

Read More

Topics: Immunoprecipitation, mCherry, Immunofluorescence, VHH, Nanobody, mNeonGreen, GFP, GFP Nanobody, GFP Antibody, TurboGFP, EGFP, Western blot, SNAP

Working with green fluorescent proteins: Tools and properties

Posted by Christoph Eckert on Feb 7, 2019 1:18:14 PM

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.

Read More

Topics: Nanobody, mNeonGreen, mNeonGreen immunoprecipitation, GFP, GFP Immunoprecipitation, TurboGFP, TurboGFP immunoprecipitation, EGFP

The best anti-GFP antibody for immunoprecipitation: GFP-Trap

Posted by Christoph Eckert on Sep 7, 2017 1:05:34 PM

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

Read More

Topics: GFP-Trap, GFP, GFP Immunoprecipitation, Best GFP antibody for IP, Best antibody for immunoprecipitation, GFP VHH, GFP Nanobody

mNeonGreen: A green fluorescent protein that is not a GFP variant

Posted by Christoph Eckert on Jul 19, 2017 8:20:46 AM

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?

Read More

Topics: mNeonGreen, mNeonGreen Antibody, mNeonGreen Western blot, mNeonGreen VHH, mNeonGreen immunoprecipitation, mNeonGreen immunofluorescence, GFP, mNeonGreen sequence

New limit in protein complex stability: GFP-binding protein:GFP complex

Posted by Klaus Herick on Feb 3, 2017 3:56:34 PM

Although traditional IgG antibodies are often used for immunoprecipitation and protein interaction analysis, GFP-binding protein (GBP, ChromoTek gt-250) and other ChromoTek VHHs (https://www.chromotek.com/about-us/the-alpaca-antibody-advantage/) easily outcompete those under challenging conditions such as elevated temperatures, varying pH or high denaturant concentrations. In fact, GBP tightly binds GFP-fusion proteins even in 8 M urea at 51 °C, which sets a new limit in protein complex stability, virtually unrivalled by any other capture molecule protein tag pair.

Read More

Topics: GFP-Trap, Nano-Trap, GFP, GFP- Binding Protein

News about Nanobodies

For researchers interested in

  • New Nanobody based tools
  • Tips and tricks
  • Featured publications

Subscribe Here!

Recent Posts

Posts by Tag

See all