GFP-Trap® A (coupled to Agarose beads)

Application: Immunoprecipitation (IP)

Catalog no.: ABIN509397

Additional Information

Components: GFP-Trap®_A (bead size: ~ 80 μm) in 20% EtOH

Description: Green fluorescent proteins (GFP) and variants thereof are widely used to study protein localization and dynamics. For biochemical analyses including mass spectroscopy and enzyme activity measurements these GFP-fusion proteins and their interacting factors can be isolated fast and efficiently (one step) via Immunoprecipitation using the GFP-Trap®. The GFP-Trap®_A enables purification of any protein of interest fused to GFP.

Characteristics: For the immunoprecipitation of GFP-fusion proteins from cellular extracts

Application Details

Protocol: For one immunoprecipitation reaction resuspend cell pellet (~107 cells) in 200 µl lysis buffer by pipetting (or using a syringe). Place the tube on ice for 30 min with extensively pipetting every 10 min. Spin cell lysate at 20.000x g for 5 -10 minutes at 4°C. Transfer supernatant to a precooled tube. Adjust volume with dilution buffer to 500 µl – 1000 µl. Discard pellet. The cell lysate can be frozen at this point for long-term storage at minus 80°C. Discard pellet. For immunoblot analysis dilute 50 µl cell lysate with 50 µl 4x SDS-sample buffer (-> refer as input). Equilibrate GFP-Trap® beads in dilution buffer. Resuspend 20 - 30 µl Beads Slurry in 500 µl ice cold dilution buffer and spin down at 2700x g for 2 minutes at 4°C. Discard supernatant and wash binder two more times with 500 µl ice cold dilution buffer.. Add cell lysate to equilibrated GFP-Trap®_A beads. Incubate with gentle end-over-end mixing for 10 min – 2 h at room temperature or 4°C. Spin tube at 2000x g for 2 minutes at 4°C. For western blot analysis dilute 50 µl supernatant with 50 µl 4x SDS-sample buffer (-> refer as non-bound). Discard remaining supernatant. Wash pellet two times with 500 µl ice cold dilution buffer (optional: increase salt concentration in the second washing step up to 500 mM). Resuspend GFP-Trap®_A beads in 100 µl 2x SDS-Sample buffer. Boil resuspended beads for 10 minutes at 95°C to dissociate the immunocomplexes from the beads. The beads can be collected by centrifugation at 2700x g for 2 minutes at 4°C and SDS-PAGE is performed with the supernatant. (-> refer as bound). (optional) elute bound proteins by adding 50 µl 0.1 M glycine pH 2.5 (incubation time: 30 sec – 2 min) followed by neutralisation with 5 µl 1M Tris-base.
Suggested Buffers (as tested in our laboratory): Lysis-buffer (native): 10 mM Tris/Cl, pH 7.5
150 mM NaCl
0.5 mM EDTA
0.5% NP40
1 mM PMSF freshly added (optional)
1x mammalian Protease Inhibitor Cocktail (e.g. Serva®) freshly added
(optional for nuclear proteins / chromatin proteins: DNaseI final conc. 1 µg/µl 2.5 mM MgCl2). Dilution-buffer 10 mM Tris/Cl, pH 7.5
150 mM NaCl
0.5 mM EDTA
1 mM PMSF freshly added (optional)
1x Protease Inhibitor Cocktail (e.g. Serva) freshly added. Wash-buffer 10 mM Tris/Cl pH 7.5
150 - 500 mM NaCl
0.5 mM EDTA
1 mM PMSF freshly added (optional)
1x Protease Inhibitor Cocktail (e.g. Serva®) freshly added. RIPA-Buffer (for cell lysis): 10 mM Tris/Cl, pH 7.5
150 mM NaCl
0.1% SDS
1% TX100
1% Deoxycholate
5 mM EDTA
1 mM PMSF freshly added (optional)
1x Protease Inhibitor Cocktail (e.g. Serva®) freshly added.

Concentration: 500 µL resin

Storage: Store material at 2-8°C, do not freeze.

Research Area: Tags/Labels

Restrictions: For Research Use only

Images

Left (IP): Pulldown of GFP with GFP-Trap®_A and GFP-Trap®_M from 293T cell extracts. Input (I) and bound (B) fractions were separated by SDS-PAGE followed by Coomassie staining. Right (Co-IP): Pulldown of GFP-PCNA with GFP-Trap®_A and GFP-Trap®_M from 293T cell extracts. Other bands: potential interaction partners of PCNA.

Publications

Rothbauer, Zolghadr, Tillib et al.: "Targeting and tracing antigens in live cells with fluorescent nanobodies." in: Nature methods, Vol. 3, Issue 11, pp. 887-9, 2006 (PubMed).

Agarwal, Hardt, Brero et al.: "MeCP2 interacts with HP1 and modulates its heterochromatin association during myogenic differentiation." in: Nucleic acids research, Vol. 35, Issue 16, pp. 5402-8, 2007 (PubMed).

Rothbauer, Zolghadr, Muyldermans et al.: "A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins." in: Molecular & cellular proteomics : MCP, Vol. 7, Issue 2, pp. 282-9, 2008 (PubMed).

Trinkle-Mulcahy, Boulon, Lam et al.: "Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes." in: The Journal of cell biology, Vol. 183, Issue 2, pp. 223-39, 2008 (PubMed).

Maerz, Dettmann, Seiler: "Hydrophobic Motif Phosphorylation Coordinates Activity and Polar Localization of the Neurospora crassa Nuclear Dbf2-Related Kinase COT1." in: Molecular and cellular biology, Vol. 32, Issue 11, pp. 2083-98, 2012 (PubMed).

Tan, Gunaratne, Lai et al.: "HPV-18 E2^E4 chimera: 2 new spliced transcripts and proteins induced by keratinocyte differentiation." in: Virology, Vol. 429, Issue 1, pp. 47-56, 2012 (PubMed).

Cho, Stevens, Sieverman et al.: "A ventrally localized protease in the Drosophila egg controls embryo dorsoventral polarity." in: Current biology : CB, Vol. 22, Issue 11, pp. 1013-8, 2012 (PubMed).

Wu, Tran, Teng et al.: "The interactome of the human respiratory syncytial virus NS1 protein interactome highlights multiple effects on host cell biology." in: Journal of virology, 2012 (PubMed).

Park, Touihri, Müller et al.: "Sec1/Munc18 protein stabilizes fusion-competent syntaxin for membrane fusion in Arabidopsis cytokinesis." in: Developmental cell, Vol. 22, Issue 5, pp. 989-1000, 2012 (PubMed).

Okada, Uezu, Soderblom et al.: "Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach." in: PLoS ONE, Vol. 7, Issue 5, pp. e37035, 2012 (PubMed).

Product: Gunaratne, Goh, Swa et al.: "Protein interactions of phosphatase and tensin homologue (PTEN) and its cancer-associated G20E mutant compared by using stable isotope labeling by amino acids in cell culture-based parallel affinity purification." in: The Journal of biological chemistry, Vol. 286, Issue 20, pp. 18093-103, 2011 (PubMed). Further details: We performed one-step purification using GFP-trap

Dosil: "Ribosome synthesis-unrelated functions of the preribosomal factor rrp12 in cell cycle progression and the DNA damage response." in: Molecular and cellular biology, Vol. 31, Issue 12, pp. 2422-38, 2011 (PubMed).

Dosko?ilova?, Pla?hal, Volc et al.: "A nodulin/glutamine synthetase-like fusion protein is implicated in the regulation of root morphogenesis and in signalling triggered by flagellin." in: Planta, 2011 (PubMed). Further details: immunoprecipitation of the protein from S27 using GFP-Trap A

Walkey, Luo, Borchers et al.: "The Saccharomyces cerevisiae fermentation stress response protein Igd1p/Yfr017p regulates glycogen levels by inhibiting the glycogen debranching enzyme." in: FEMS yeast research, 2011 (PubMed).

Meruvu, Hugendubler, Mueller: "Regulation of Adipocyte Differentiation by the Zinc Finger Protein ZNF638." in: The Journal of biological chemistry, 2011 (PubMed). Further details: For GFP IP assays, transfected HEK-293 cells were lysed, 30 ?l of GFP-Trap

Nagel, Albrecht, Milovic-Holm et al.: "Herpes Simplex Virus Immediate Early Protein ICP0 is Targeted by SIAH-1 for Proteasomal Degradation." in: Journal of virology, 2011 (PubMed).

García-Gómez, Lebaron, Froment et al.: "Dynamics of the putative RNA helicase Spb4 during ribosome assembly in Saccharomyces cerevisiae." in: Molecular and cellular biology, 2011 (PubMed).

Engeland, Oberwinkler, Schümann et al.: "The cellular protein Lyric interacts with HIV-1 Gag." in: Journal of virology, 2011 (PubMed).

Doggett, Zhao, Mork et al.: "Phosphorylation of LRRK2 serines 955 and 973 is disrupted by Parkinson's disease mutations and LRRK2 pharmacological inhibition." in: Journal of neurochemistry, 2011 (PubMed).

Essid, Gopaldass, Yoshida et al.: "Rab8a regulates the exocyst-mediated kiss-and-run discharge of the Dictyostelium contractile vacuole." in: Molecular biology of the cell, 2012 (PubMed).

Moulin, Anderton, Voss et al.: "IAPs limit activation of RIP kinases by TNF receptor 1 during development." in: The EMBO journal, 2012 (PubMed). Further details: GFP fusion proteins were precipitated using GFP

Poulsen, Madsen, Young et al.: "Comprehensive profiling of proteome changes upon sequential deletion of deubiquitylating enzymes." in: Journal of proteomics, Vol. 75, Issue 13, pp. 3886-97, 2012 (PubMed).

Steenhuis, Froemming, Reinheckel et al.: "Proteolytic cleavage of the disease-related lysosomal membrane glycoprotein CLN7." in: Biochimica et biophysica acta, Vol. 1822, Issue 10, pp. 1617-28, 2012 (PubMed).

Tomaštíková, Cenklová, Kohoutová et al.: "Interactions of an Arabidopsis RanBPM homologue with LisH-CTLH domain proteins revealed high conservation of CTLH complexes in eukaryotes." in: BMC plant biology, Vol. 12, pp. 83, 2012 (PubMed).

Alves-Silva, Sánchez-Soriano, Beaven et al.: "Spectraplakins promote microtubule-mediated axonal growth by functioning as structural microtubule-associated proteins and EB1-dependent +TIPs (tip interacting proteins)." in: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 32, Issue 27, pp. 9143-58, 2012 (PubMed).

Chen, Schaap: "The prokaryote messenger c-di-GMP triggers stalk cell differentiation in Dictyostelium." in: Nature, Vol. 488, Issue 7413, pp. 680-3, 2012 (PubMed).

Kraft, Kijanska, Kalie et al.: "Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy." in: The EMBO journal, Vol. 31, Issue 18, pp. 3691-703, 2012 (PubMed).

Castro-Castro, Janke, Montagnac et al.: "ATAT1/MEC-17 acetyltransferase and HDAC6 deacetylase control a balance of acetylation of alpha-tubulin and cortactin and regulate MT1-MMP trafficking and breast tumor cell invasion." in: European journal of cell biology, 2012 (PubMed).

Buisson, Masson: "PALB2 self-interaction controls homologous recombination." in: Nucleic acids research, 2012 (PubMed).

Hermanson, Carlson, Riddle et al.: "Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation." in: PLoS ONE, Vol. 7, Issue 8, pp. e43580, 2012 (PubMed).

Maemoto, Kiso, Shibata et al.: "Analysis of limited proteolytic activity of calpain-7 using non-physiological substrates in mammalian cells." in: The FEBS journal, 2013 (PubMed).

Elias, Wu, Chen: "Tumor Suppressor Protein p53 Negatively Regulates Human Pregnane X Receptor Activity." in: Molecular pharmacology, 2013 (PubMed).

Stam, Jupe, Howden et al.: "Identification and Characterisation CRN Effectors in Phytophthora capsici Shows Modularity and Functional Diversity." in: PLoS ONE, Vol. 8, Issue 3, pp. e59517, 2013 (PubMed).

Convertini, Zhang, de la Grange et al.: "Genome wide array analysis indicates that an amyotrophic lateral sclerosis mutation of FUS causes an early increase of CAMK2N2 in vitro." in: Biochimica et biophysica acta, 2013 (PubMed).

Moreno, Shyu, Campos et al.: "Negative Feedback Control of Jasmonate Signaling by an Alternative Splice Variant of JAZ10." in: Plant physiology, 2013 (PubMed).

Ono, Yamada, Endo et al.: "Analysis of Human Protein Replacement Stable Cell Lines Established using snoMEN-PR Vector." in: PLoS ONE, Vol. 8, Issue 4, pp. e62305, 2013 (PubMed).

Stockert, Wolf, Kaddatz et al.: "Regulation of TAK1/TAB1-Mediated IL-1β Signaling by Cytoplasmic PPARβ/δ." in: PLoS ONE, Vol. 8, Issue 4, pp. e63011, 2013 (PubMed).