GFP-Trap® M (coupled to magentic particles)

Application: Immunoprecipitation (IP)

Catalog no.: ABIN509401

Additional Information

Components: Magnetic GFP-Trap®-M (size ~ 0.5 -1 μM in PBS 0.1% BSA )

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).

Product: Mitchell, Lau, Lambert et al.: "Regulation of septin dynamics by the Saccharomyces cerevisiae lysine acetyltransferase NuA4." in: PLoS ONE, Vol. 6, Issue 10, pp. e25336, 2011 (PubMed).

Jovic, Kean, Szentpetery et al.: "TWO PI 4-KINASES CONTROL LYSOSOMAL DELIVERY OF THE GAUCHER DISEASE ENZYME, β-GLUCOCEREBROSIDASE." in: Molecular biology of the cell, 2012 (PubMed).

Nakatogawa, Ohbayashi, Sakoh-Nakatogawa et al.: "The Autophagy-related Protein Kinase Atg1 Interacts with the Ubiquitin-like Protein Atg8 via the Atg8 Family Interacting Motif to Facilitate Autophagosome Formation." in: The Journal of biological chemistry, Vol. 287, Issue 34, pp. 28503-7, 2012 (PubMed).

Watanabe, Kobayashi, Yamamoto et al.: "Structure-based analyses reveal distinct binding sites for Atg2 and phosphoinositides in Atg18." in: The Journal of biological chemistry, 2012 (PubMed).

Hsieh, Cheng, Lin: "Functional characterization of an abiotic stress-inducible transcription factor AtERF53 in Arabidopsis thaliana." in: Plant molecular biology, 2013 (PubMed).

Cheng, Tsai, Huang et al.: "Ser/Thr Kinase-Like Protein of Nicotiana benthamiana Is Involved in the Cell-to-Cell Movement of Bamboo mosaic virus." in: PLoS ONE, Vol. 8, Issue 4, pp. e62907, 2013 (PubMed).