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借助Chromotek公司GFP-Trap如何设计成功的IP实验？ How to plan an immunoprecipitation of your GFP-fusion protein when using the ChromoTek GFP-Trap® Preamble This document provides practical information on how to apply the ChromoTek GFP-Trap® for immunoprecipitation. Introduction The ChromoTek GFP-Trap® is based on a GFP-binding protein derived from an Alpaca single variable domain antibody, also called VHH or nanobody (figure 1). The GFP-Trap has particular properties and provides some advantages over traditional IgG antibodies when applied in immunoprecipitations. Planning of the experiment There are some experimental aspects that you should consider when planning immunoprecipitation of your GFP-fusion protein of interest using the ChromoTek GFP-Trap®. Below, we guide you through each step. There are 3 matrices available: Feature Agarose beads Magnetic agarose beads Magnetic beads Matrix Agarose, 4% highly cross-linked Agarose, 6% cross-linked Silica, non-porous Partical size 45-165μm 20-40μm 0.5-1μm Binding capacity 2-3μg per 10μl bead slurry 2-3μg per 10μl bead slurry 0.5μg per 10μl bead slurry Magnetic no yes yes Pre-clearing: Pre-clearing is an optional step to remove proteins or DNA which bind non-specifically to the solid-phase support. It includes the incubation of the cell extract with plain beads (e.g.binding control agarose beads) before performing the actual immunoprecipitation experiment with the GFP-Trap. After successful pre-clearing, non-specific proteins or other components will not be co-purified with the protein of interest. CRAPome: By nature, every matrix and binding molecule may nonspecifically bind some proteins resulting in protein background. Scientists have established the internet-based database CRAPome at www.crapome.org. This database stores and annotates negative controls generated by the proteomics research community. CRAPome helps to determine the background contaminants--for example, proteins that interact with the solid-phase support,affinity reagent or epitope tag. Specificity - What fluorescent proteins are captured by the GFP-Trap® The GFP-Trap® specifically binds to most of the common GFP derivatives: Fluorescent protein GFP-Trap®   Fluorescent protein GFP-Trap® (e)GFP √ (e)Citrine √ tagGFP2 √ AcGFP √ turboGFP - Superfolder GFP √ mClover √ mCerulean √ YFP √ pHluorin √ CFP √ GFP S65T √ Venus √ - - The ChromoTek GFP-Trap® only binds properly folded active GFP. It is believed that this is because that nanobody binds to a three-dimensional epitope of GFP. The nanobody’s elongated CDR3 (complementarity determining region 3) allows to reach into clefts and enzymatic centers of proteins, which are not accessible to conventional antibodies but results in very strong binding and very low dissociation constants of this GFP nanobody. Therefore that anti-GFP nanobody is not suitable for protein detection in Western Blots. For Western Blot detection of GFP (fusion proteins) ChromoTek recommends the traditional antibody anti-GFP antibody 3H9 (rat monoclonal, see complementary products). Controls – What controls should I conduct to validate the experimental data? Below find some suggestions by application: For Immunoprecipitation (IP):      • GFP-Trap® for IP of GFP-fusions and a non-relevant Nano-Trap as negative control, e.g. Myc-Trap®, GST-Trap or MBP-Trap For Co-Immunoprecipitation (Co-IP) of protein complex AB: Cell Lysis – What to consider when preparing a cell lysate? Lysis buffers:      • A non-denaturing lysis buffer is suitable for Co-IP, because proteins will remain in their native conformation      • The RIPA (Radio Immunoprecipitation Assay) buffer might denature proteins or disrupt protein complexes Inhibitors:      • Add protease inhibitors to prevent proteolysis!      • Preserve posttranslational modifications of your protein and add e.g. phosphatase inhibitors!      • Prevent degradation of your protein by keeping your samples on ice! Immunoprecipitation – Binding of the GFP-fusion Since the GFP binding protein is covalently coupled to the beads’ surface, the GFP-Trap®beads are ready-to-use and can be directly added to the prepared lysate. The affinity ofGFP-nanobody is in the picomolar range, therefore depletion of GFP-fusions can becompleted within 5-30 minutes. Buffer compatibility of the GFP-Trap® for binding and washing The GFP-Trap® is compatible with most wash buffers and stable under harsh conditions including:      • Up to 1 M NaCl and 8 M Urea      • Up to 0.2% SDS and 2% NP-40 Elution strategies The elution of the bound GFP-fusion protein by a competitive peptide, which replaces the GFP-fusion protein doesn’t work. Also, the addition of chaotropic compounds like urea don’telute the bond GFP-fusion protein as the GFP-Trap® works under denaturing conditions. We therefore recommend to elute with:      • SDS, e.g. SDS sample buffer, is a very effective way to elute the bound GFP-tagged protein. The elution results in denatured GFP-fusions.      • 0.2 M glycine pH 2.5        Alternatively you may elute with glycine at pH 2.5. It is recommended to repeat this elution step as the pH shift elution works incompletely. The repetition will improve the elution efficiency.        Very important: Don’t forget to neutralize proteins immediately after elution! As an alternative to above elution options, a protease cleavage site between GFP and the fusion protein can be introduced. This option is recommended if less stable proteins have been bound or if you want to enrich your native protein of interest. Furthermore, consider whether you really need to elute the bound protein of interest from the beads rather than conduct the downstream analysis “on-bead”:      • Proteins can be digested when still coupled to the beads for subsequent mass spectrometry analysis.      • Enzymatic activity assays can be performed when still coupled to the beads if the active center is not blocked. Reproducibility The GFP-Trap® is a small, soluble and stable single polypeptide chain that is recombinantly expressed in bacteria. This in combination with quality control makes its production robust and reproducible for reliable results. Selected References to introduce Nanobodies and their applications: Nanobodies as probes for protein dynamics in vitro and in cells Dmitriev, O. Y., Lutsenko, S. and Muyldermans, S. in: Journal of Biological Chemistry, 2015 – jbc-R115. A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins Rothbauer, U., Zolghadr, K., Muyldermans, S., Schepers, A., Cardoso, M. C., Leonhardt, H. in: Mol Cell Proteomics, 2008 Feb;7(2):282-9. Epub 2007 Oct 21 Nanobody-based products as research and diagnostic tools De Meyer, T., Muyldermans,S. and Depicker, A. in: Trends in Biotechnology, 2014 May; 32 (5): 263-270; Beneficial properties of single-domain antibody fragments for application in immunoaffinity purification and immuno-perfusion chromatography Verheesen P., Ten Haaft M. R., Lindner N., Verrips C.T., de Haard J. J. W. in: Biochim. Biophys. Acta, 2003; 1624(1–3): 21–28 A highly specific gold nanoprobe for live-cell single-molecule imaging Leduc, C., Si, S., Gautier, J., Soto-Ribeiro, M., Wehrle-Haller, B., Gautreau, A., .Giannone, G., Cognet, L. & Lounis, B. in: Nano letters, 2013: 13(4), 1489-1494. Nanobody-based chromatin immunoprecipitation. Duc, T. N., Hassanzadeh-Ghassabeh, G., Saerens, D., Peeters, E., Charlier, D., & Muyldermans, S. in: Single Domain Antibodies: Methods and Protocols, 2013: 491-505       Product name Size Code       GFP-Trap® A        ▶coupled to agarose beads 10 rxns(250µl resin) gta-10 20 rxns(500µl resin) gta-20 100 rxns(2.5ml resin) gta-100 200 rxns(5ml resin) gta-200 400 rxns(10ml resin) gta-400       GFP-Trap® A Kit        ▶GFP-Trap® A        ▶incl.lysis wash and elution buffers 20 rxns(500µl resin) gtak-20       GFP-Trap® MA         ▶coupled to magnetic agarose beads 10 rxns(250µl resin) gtma-10 20 rxns(500µl resin) gtma-20 100 rxns(2.5ml resin) gtma-100 200 rxns(5ml resin) gtma-200 400 rxns(10ml resin) gtma-400       GFP-Trap® MA Kit        ▶GFP-Trap® MA        ▶incl.lysis,wash and elution buffers 20 rxns(500µl resin) gtmak-20       GFP-Trap® M        ▶coupled to magnetic particles 10 rxns(250µl resin) gtm-10 20 rxns(500µl resin) gtm-20 100 rxns(2.5ml resin) gtm-100 200 rxns(5ml resin) gtm-200 400 rxns(10ml resin) gtm-400       GFP-Trap® M Kit         ▶GFP-Trap® M         ▶incl.lysis,wash and elution buffers 20 rxns(500µl resin) gtmk-20       GFP- multi Trap®         ▶black 96 well plate 96 rxns(1 plate) gtp-96 480 rxns(5 plate) gtp-480       GFP-Trap®         ▶uncoupled protein 250µl gt-250       Spin columns 10 units sct-10 20 units sct-20 50 units sct-50       Binding control         ▶agarose beads 500µl(20 rxns) bab-20       Binding control         ▶magnetic agarose beads 500µl(20 rxns) bmab-20       Binding control         ▶magnetic particles 500µl(20 rxns) bmp-20       GFP antibody(3H9)         ▶rat monocional 100µl 3H9 产品咨询热线：4006-400-850 Email：info@moreybio.com QQ：2885367975