Description The ChromoTek GFP-Trap® Magnetic Agarose are affinity beads for immunoprecipitation of GFP-fusion proteins. It comprises a GFP Nanobody/ VHH coupled to magnetic agarose beads.Specificity GFP, EGFP, CFP, YFP, BFP and many more derivatives, see: Fluorescent protein specificity tableApplications Immunoprecipitation/ Co-IP Mass spectrometry On-bead enzyme assays ChIP, RIP analysis AffinityDissociation constant KD of 1 pMWash buffer compatibility 1 mM DTT, 3 M Guanidinium•HCl, 8 M Urea, 2 M NaCl, 2 % Nonidet P40 Substitute, 1 % SDS, 1 % Triton X-100Specificity (selection)- AcGFP, Clover, eGFP, Emerald, GFP, GFP5, GFP Envy, GFP S65T, mGFP, mPhluorin, PA-GFP, Superfolder GFP, TagGFP, TagGFP2, monomeric eGFP A206K - CFP - YFP, Citrine, eCitrine, eYFP, Venus, Ypet - BFP For complete list, please click here: Fluorescent protein specificity tableBinding capacity 10 µL slurry bind more than 8 µg of recombinant GFPCoupled Nanobody/ VHH Recombinant, monoclonal anti-Green Fluorescent Protein (GFP) single domain antibody (sdAb) fragmentBead properties Bead size: ~ 40 µm Storage buffer: 20 % EtOHElution - SDS sample buffer - 0.2 M glycine pH 2.5 Instead of elution, we recommend on-bead assays like on-bead digestion for MS analysis.Compatibility with mass spectrometry The GFP-Trap is optimized for on-bead digestion. Complete tryptic digest results in 4-5 peptidesRRID AB_2631358Storage instructions Shipped at ambient temperature. Upon receipt store at 4°C; stable for one year. Laura Trinkle-Mulcahy, Séverine Boulon, Yun Wah Lam , Roby Urcia , François-Michel Boisvert , Franck Vandermoere, Nick A. Morrice, Sam Swift, Ulrich Rothbauer, Heinrich Leonhardt, Angus Lamond. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J. Cell Biol. Vol. 183 No. 2 223–239Zoltan Lipinszki, Peng Wang, Rhys Grant, Catherine Lindon, Nikola S. Dzhindzhev, Pier Paolo D’Avino, Marcin R. Przewloka, David M. Glover, Vincent Archambault; Affinity Purification of Protein Complexes from Drosophila Embryos in Cell Cycle Studies. Methods Mol Biol. 2014;1170:571-88. doi: 10.1007/978-1-4939-0888-2_33.Arne H. Smits, Pascal W. T. C. Jansen, Ina Poser, Anthony A. Hyman, Michiel Vermeulen; Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics. Nucleic Acids Res 2013; 41 (1): e28. doi: 10.1093/nar/gks941Benedetta Turriziani, Amaya Garcia-Munoz, Ruth Pilkington, Cinzia Raso, Walter Kolch, Alexander von Kriegsheim; On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics. Biology 2014, 3(2), 320-332; doi:10.3390/biology3020320David R. Croucher, Mary Iconomou, Jordan F. Hastings, Sean P. Kennedy, Jeremy Z. R. Han, Robert F. Shearer, Jessie McKenna, Adrian Wan, Joseph Lau, Samuel Aparicio, Darren N. Saunders; Bimolecular complementation affinity purification (BiCAP) reveals dimer-specific protein interactions for ERBB2 dimers. Sci. Signal. 12 Jul 2016 : ra69.Timothy D. Cummins and Gopal P. Sapkota. Characterization of protein complexes using chemical cross-linking coupled electrospray mass spectrometry. ArXiV 2016 arxiv.org/ftp/arxiv/papers/1606/1606.04247.pdfJennifer N. Byrum, Shuying Zhao, Negar S. Rahman, Lori M. Gwyn,William Rodgers, and Karla K. Rodgers An Interdomain boundary in RAG1 facilitates cooperative binding to RAG2 in formation of the V(D)J recombinase complex Julian R. Avila,a,b Jin Suk Lee,a,b and Keiko U. Torii Co-Immunoprecipitation of Membrane-Bound Receptors Is there a difference in binding when I use the N-terminal vs. C-terminal GFP-fusions? The GFP-Trap®has a slightly higher affinity for C-terminal GFP-fusions. You can compensate this by an elongated incubation time ( 1 - 2 h instead of 15 – 30 min). You may try to elute with free GFP. However, please be aware that this method will not quantitatively elute your fusion protein of interest. No, the GFP-Trap doesn\'t bind TurboGFP. TurboGFP is a green fluorescent protein derived from CopGFP of the copepod Pontellina plumata whereas GFP has been originally isolated from jellyfish Aequorea Victoria. TurboGFP shares only ~20 % sequence identity with the commonly used GFP variants. Can I purify GFP labeled fusion proteins directly from tissue samples, i.e. in a denaturing buffer? In principle the GFP-Trap®is very stable even under harsh buffer conditions (e.g. RIPA buffer containing 0.1% SDS or 1 M urea). Will the eluted GFP binding protein cross react with a secondary Ig specific antibody? Since the binding protein used in the GFP-Trap®does not have any significant homology with goat, mouse, rat or human antibodies, unspecific reactions with a secondary Ig specific antibody should not occur. GFP-TrapAgarose and GFP-TrapMagnetic Agarose usually bind around 8 µg GFP per 10 µL slurry,GFP-Trap Magnetic Beadsbinds around 1 µg per 10 µL slurry. For preclearing of your sample we recommend to use our binding controls (bab-20 or bmab-20) when you use GFP-Trap Agarose or Magnetic Agarose for the IP.Please find more information in our Troubleshooting guide. You can elute your fusion protein of interest with 0.2 M glycine pH 2.5 at room temperature. Pipette the beads up and down for 60-120 seconds and repeat this step. Ensure to neutralize your supernatant immediately afterwards by adding 1 M Tris base pH 10.4 For one immunoprecipitation reaction, we recommend using ~10^6 - 10^7 mammalian cells. The yield is also dependent on the expression level of your protein of interest and the interaction partners. For other type of cells than mammalian cells, we recommend using 0.5 - 1.0 mg of cell extract. No, you can directly conduct an on-bead digestion after immunoprecipitation. This procedure allows faster and more efficient sample preparation and a potential higher yield. Please find more information here:Preomics kitProtocol on-bead digestion No, you can directly perform your enzymatic assay on the beads if the active center is not blocked.Please find more information in our application note enzymatic activity assay Generally heavy chain antibodies do have high affinities to their antigens with dissociation constants in the low nanomolar down to the picomolar range. ChromoTek has determined the following KD values: GFP-Trap: 1 pM, picomolar (10-12 molar)* RFP-Trap: 5 nM, nanomolar (10-9 molar) MBP-Trap: 4 nM, nanomolar (10-9 molar) GST-Trap: 1 nM, nanomolar (10-9 molar)* Myc-Trap (with 2x Myc peptide): 0.5 nM, nanomolar (10-9) Spot-Trap: 6 nM, nanomolar (10-9 molar) *Kinetic parameter has been measured using the switchSENSE technology using electro-switchable nanolevers to analyze molecular interactions. switchSENSE is a proprietary technology from Dynamic Biosensors (www.dynamic-biosensors.com). 25 µL slurry are sufficient for one pull-down reaction as the affinity of the traps is very high.GFP-TrapAgarose Magnetic Agarose Magnetic Beads Matrix Agarose (4% cross-linked) Magnetic agarose (6% cross linked) Magnetic Beads M-270 Bead form Porous Porous; sold iron core Solid Ligand GFP VHH GFP VHH GFP VHH GFP-tagged protein size* Small to large size Small to large size Small to very large size; no size limitation Color White Black Brown Medium particle size 90 µm 40 µm 2.8 µm Binding capacity 12 µg/ 10 µL 8 µg/ 10 µL 1 μg/ 10 μL Background Very low Low Low Magnetic separation automation No Yes Yes May be centrifuged up to 2,500 x g 800 x g 8,000 x g * Does depend on protein size and shape, protein multimers, complexes and interaction partners The Magnetic Beads matrix is inert and shouldn’t bind background proteins. Hence, unconjugated Magnetic Beads shouldn’t be used for preclearing. To investigate unspecific binding to GFP-Trap Magnetic Beads, we recommend to perform the IP with mock cell lysate without GFP-fusion or with GFP only. Effective immunoprecipitation of GFP-fusion proteins for reproducible results short incubation (5-30 min), no heavy light antibody chains Extraordinary stable reliable binding High affinity (KD of 1pM*) to bind even proteins at expressed at low levels Gold standard with more than 1.500 publications The GFP-Trap Magnetic Agarose is also available in a kit, including: GFP-Trap Magnetic Agarose lysis*, wash and elution buffers *The lysis buffer provided is suitable for mammalian cells. For other cell types use another suitable lysis buffer. control for unspecific binding of proteins, DNA, etc. to beads pre-clearing of cell lysate Binding Control for GFP-Trap Magnetic Agarose GFP-Trap Agarose, when lowest background and high binding capacity IP is needed. GFP-Trap Magnetic Agarose, when magnetic separation and high binding capacity IP is needed. GFP-Trap Magnetic Beads, when very large proteins/complexes are investigated, and magnetic separation is needed for IP. \"Your GFP-Trap® is great! I have never seen such efficient reagent for pull down.Prof. Dr. Tomoyuki Tanaka, Wellcome Trust Centre for Gene Regulation Expression, University of Dundee \"We recently had excellent MS results with the GFP-Trap®.” Dr. Gwyneth Ingram, IMPS, Edinburgh \"We have been pleased with the results from both of the products: GFP-Trap_A®and GFP-Trap_M®” Katharine S. Ullman, Ph.D., Assoc. Professor, Huntsman Cancer Institute, University of Utah\"I just wanted to give you a quick update on our experiences testing your GFP-Trap®. This will be quick because our experience is that they work fantastically well!” Prof. Dr. A.I. Lamond, Wellcome Trust Center, University of Dundee\"We were gob smacked when we did our own IP and saw the glowing green GFP-Trap®beads...” Prof. Dr. Laura Trinkle-Mulcahy, University of Ottawa\"The GFP-Trap® worked very nicely to pull-down our actin binding protein...” Prof. Dr. Evelyne Friederich, University of Luxembourg\"I am brim over with enthusiasm for your GFP-Trap®; great product and great idea; Hats off!” Dr. Monika Jedrusik-Bode, MPI for Biophysical Chemistry, Göttingen GFP-Trap®is the best beads ever! Dr. Kenkyo Matsuura, Department of Pharmacology and Cancer Biology, Duke University\"When I used antibody for immunoprecipitation, strong background was usually hard trouble. I think that GFP-Trap®is revolution.\" Takeshi Mizuno, Ph. D., Advanced Science Institute, RIKEN (Institute of Physical and Chemical Research) The results obtained with the GFP-Trap®are much more clean (…) I am thus very happy about the GFP-Trap_A®product!” Florence Besse, PhD, Univ. Nice Sophia-Antipolis\"Man erlebt es in der Wissenschaft ja eher selten, dass ein neues Tool in den eigenen Händen genau so gut funktioniert, wie man es sich erhofft hatte - mit der GFP-Trap_A® war das bei mir der Fall! Das hat mich wirklich überzeugt!” Friederike Althoff, Institut of Zoology, University of Zürich\"The beads GFP-Trap®_M are fantastic!” Francesca Sicilia, Dipartimento di Biologia Vegetale, Universitá di Roma La Sapienza Your GFP-Trap® is such a powerful tool for bio-sensing applications! Stable, specific, sensitive, soluble...it has all the ideal features of a capturing molecule. And it comes at a verycompetitive price.Dr. Eduardo Della Pia, Nano Science Center, University of Copenhagen