ChromoBlog

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/).

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

A collaboration between ChromoTek GmbH and Ludwig-Maximilians-University Munich (Germany) has yielded a novel nanobody that sheds new light on an important protein complex called INO80. This work was recently published in the high-impact journal Nature Structural & Molecular Biology (Knoll et al. 2018).

Topics: Immunoprecipitation, Nanobody, Crystallisation

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.

Topics: Immunoprecipitation, TurboGFP, TurboGFP immunoprecipitation, Best TurboGFP antibody, TurboGFP Nanobody

Multiple literature references for GST-Trap

This article provides an overview of scientific publications that reference the ChromoTek GST-Trap superior binding performance for various applications. These include immunoprecipitation (IP), Co-IP of Glutathione S-Transferase (GST)-fusion proteins, and Luminex bead assays of GST-tagged and GFP-tagged proteins. Besides, usages for depletion of GST (glutathione sepharose)  after cleavage from recombinant fusion proteins and affinity purification of GST-tagged proteins are possible.

Topics: Immunoprecipitation, GST, affinity resin, GST immunoprecipitation, references, affinity purification, GST pulldown

Practical considerations and instructions

Introduction

The ChromoTek Myc-Trap is an affinity resin that consists of Myc-binding protein derived from an Alpaca single domain antibody, also called anti-Myc VHH or “anti-Myc nanobody”, which is coupled to agarose and magnetic agarose beads. The Myc-Trap provides advantages over conventional IgG antibodies when applied in immunoprecipitation (IP) and affinity purification experiments:

Topics: Immunoprecipitation, Myc-tag, Myc-tag IP, single domain antibody, Myc Nanobody

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. 

Topics: Immunoprecipitation, Myc-tag, Myc-tag IP

Immunoprecipitation (IP) followed by mass spectrometry (MS) analysis is a powerful method to identify interaction partners of a protein of interest. However, sometimes it can be difficult to obtain reliable results.

Now, the combined use of ChromoTek GFP-Trap for immunoprecipitation and PreOmics iST sample preparation kit is in fact a straight forward, reproducible, and reliable approach to identify protein interaction partners. It even preserves posttranslational modification (PTM) depending protein-protein interactions.

In a recent applications note we have demonstrated how protein interaction partners of PARP1 can be identified: We have immunoprecipitated eGFP-tagged PARP1 protein and its interacting partners using the GFP-Trap_M and subsequently processed the pulldown for MS analysis following the instructions of the iST kit.

PARylation mediates the interaction of PARP1 to several DNA repair proteins. Our results show that those interactions are not disrupted by neither the IP nor the MS sample preparation used herein. Therefore, the streamlined combination of the GFP- Trap and the iST kit’s workflow proves to preserve PTM-mediated protein-protein interactions.

 

 

The PreOmics iST kit can also be used with other ChromoTek Nano-Traps.

Topics: GFP-Trap, Immunoprecipitation, Mass spec, GFP Immunoprecipitation, GFP MS

Pull-down of proteins can be difficult, particularly when they are expressed at low levels. Here we present 5 tips, which will considerably improve your IP results.

 

1. Protein concentration matters

The higher the protein concentration, the higher the IP yield. Try to use a concentration as high as possible.It makes a significant difference if the concentration of your protein during IP is 1 nM (low protein expression level), about 50 nM (intracellular endogenous protein expression level, will be diluted by buffer for IP) or 1000 nM (high protein concentration).

Topics: Nano-Trap, Immunoprecipitation, Co-IP, GST pulldown, MBP pulldown

MAP kinase-activated protein kinase 2 (MAPKAP-K2, MK2; Gene ID: 9261) is a 400 AA (46kDa) large enzyme that plays a central role mainly in the inflammatory response and cytokines production. It belongs to the serine/threonine-protein kinase family and is also involved in endocytosis, reorganization of the cytoskeleton, cell migration, cell cycle control, chromatin remodeling, DNA damage response and transcriptional regulation.^1-4

Following stress, it is phosphorylated (at Thr-222, Ser-272 and Thr-334) and activated by MAP kinase p38-alpha/MAPK14, leading to phosphorylation of substrates.⁵ Phosphorylation of Thr-334 (located between the kinase domain and the C-terminal regulatory domain) may serve as a switch for MK2 nuclear import and export. Phosphorylated MK2 masks the nuclear localization signal at its C-terminus by binding to p38. It unmasks the nuclear export signal, which is part of the second C-terminal helix packed along the surface of kinase domain C-lobe, and thereby carries p38 to the cytoplasm.^{6, 7}

Topics: Nano-Trap, MK2-Trap, Immunoprecipitation, Co-IP