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/).

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

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).

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.

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.

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:

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. 

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).