GFP-Trap® A (coupled to Agarose beads)

Antigen: Green Fluorescent Protein (GFP)

Application: Immunoprecipitation (IP)

Catalog no.: ABIN509397

Additional Information

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

Description: 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.

Application Details

Protocol:

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

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

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

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).

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