GFP-Trap® A (coupled to Agarose beads)

Antigen: Green Fluorescent Protein (GFP)

Applikation: Immunpräzipitation (IP)

Produktnummer: ABIN509400

Produktbeschreibung

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

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

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

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).

Larance, Kirkwood, Xirodimas et al.: "Characterization of MRFAP1 Turnover and Interactions Downstream of theNEDD8 Pathway." in: Molecular & cellular proteomics : MCP, 2011 (PubMed).