GFP-Trap® A (coupled to Agarose beads) Kit

Antigen: Green Fluorescent Protein (GFP)

Applikation: Immunpräzipitation (IP)

Produktnummer: ABIN509405

Produktbeschreibung

Beschreibung: Green fluorescent proteins (GFP) and variants thereof are widely used to study protein localization and dynamics. For biochemical analyses including mass pectroscopy 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.

Anwendungen

Protokoll:

1. For one immunoprecipitation reaction resuspend cell pellet (~107 cells) in 200 μl lysis buffer by pipetting (or using a syringe)
2. Place the tube on ice for 30 min with extensively pipetting every 10 min
3. Spin cell lysate at 20.000x g for 5 -10 minutes at 4°C
4. 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)
5. 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.
6. Add cell lysate to equilibrated GFP-Trap®_A beads
7. Incubate with gentle end-over-end mixing for 10 min – 2 h at room temperature or 4°C
8. Spin tube at 2000x g for 2 minutes at 4°C
9. For western blot analysis dilute 50 μl supernatant with 50 μl 4x SDS-sample buffer (-> refer as non-bound)
10. Discard remaining supernatant
11. Wash pellet two times with 500 μl ice cold dilution buffer (optional: increase salt concentration in the second washing step up to 500 mM)
12. Resuspend GFP-Trap®_A beads in 100 μl 2x SDS-Sample buffer
13. 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)
14. (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

Elution-Buffer:

0.2 M glycine, pH 2.5

Bestandteile: GFP-Trap® coupled to agarose beads, particle size ~ 80 µm, stored in 20% EtOH, Kit including lysis, wash and elution buffers

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

Forschungsgebiet: Tags/Labels

Beschränkungen: Nur für Forschungszwecke einsetzbar

Bilder

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.

Publikationen

Publikationen: 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).

Miroci, Schob, Kindler et al.: "Makorin Ring Zinc Finger Protein 1 (MKRN1), a Novel Poly(A)-binding Protein-interacting Protein, Stimulates Translation in Nerve Cells." in: The Journal of biological chemistry, Vol. 287, Issue 2, pp. 1322-34, 2012 (PubMed).

Jost, Rottach, Milden et al.: "Generation and characterization of rat and mouse monoclonal antibodies specific for MeCP2 and their use in X-inactivation studies." in: PLoS ONE, Vol. 6, Issue 11, pp. e26499, 2011 (PubMed).

Neumuller, Wirtz-Peitz, Lee et al.: "Stringent Analysis of Gene Function and Protein-Protein Interactions Using Fluorescently Tagged Genes." in: Genetics, 2011 (PubMed).

von Maltzahn, Bentzinger, Rudnicki: "Wnt7a-Fzd7 signalling directly activates the Akt/mTOR anabolic growth pathway in skeletal muscle." in: Nature cell biology, Vol. 14, Issue 2, pp. 186-91, 2012 (PubMed).

Corrotte, Fernandes, Tam et al.: "Toxin Pores Endocytosed During Plasma Membrane Repair Traffic into the Lumen of MVBs for Degradation." in: Traffic (Copenhagen, Denmark), 2012 (PubMed).

Howitt, Lackovic, Low et al.: "Ndfip1 regulates nuclear Pten import in vivo to promote neuronal survival following cerebral ischemia." in: The Journal of cell biology, Vol. 196, Issue 1, pp. 29-36, 2012 (PubMed).

OLoghlen, Muñoz-Cabello, Gaspar-Maia et al.: "MicroRNA regulation of Cbx7 mediates a switch of Polycomb orthologs during ESC differentiation." in: Cell stem cell, Vol. 10, Issue 1, pp. 33-46, 2012 (PubMed).

Weissbach, Scadden: "Tudor-SN and ADAR1 are components of cytoplasmic stress granules." in: RNA (New York, N.Y.), 2012 (PubMed).

Zwaenepoel, Naba, da Cunha et al.: "Ezrin regulates microvillus morphogenesis by promoting distinct activities of Eps8 proteins." in: Molecular biology of the cell, 2012 (PubMed).

Magyar, Horváth, Khan et al.: "Arabidopsis E2FA stimulates proliferation and endocycle separately through RBR-bound and RBR-free complexes." in: The EMBO journal, 2012 (PubMed).

Borrego-Pinto, Jegou, Osorio et al.: "Samp1 is a component of TAN lines and is required for nuclear movement." in: Journal of cell science, 2012 (PubMed).

Sala-Valdés, Gordón-Alonso, Tejera et al.: "Association of syntenin-1 with M-RIP polarizes Rac-1 activation during chemotaxis and immune interactions." in: Journal of cell science, 2012 (PubMed).

Tibarewal, Zilidis, Spinelli et al.: "PTEN Protein Phosphatase Activity Correlates with Control of Gene Expression and Invasion, a Tumor-Suppressing Phenotype, But Not with AKT Activity." in: Science signaling, Vol. 5, Issue 213, pp. ra18, 2012 (PubMed).

Guttery, Ferguson, Poulin et al.: "A Putative Homologue of CDC20/CDH1 in the Malaria Parasite Is Essential for Male Gamete Development." in: PLoS pathogens, Vol. 8, Issue 2, pp. e1002554, 2012 (PubMed). Weitere Details: GFP-tagged CDC20 proteins were then immunoprecipitated using GFP-TRAP beads

Beli, Lukashchuk, Wagner et al.: "Proteomic Investigations Reveal a Role for RNA Processing Factor THRAP3 in the DNA Damage Response." in: Molecular cell, 2012 (PubMed).