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GFP-Trap® M (coupled to magentic particles)
|10 references available|
|Quantity||20 tests (500 µL resin)|
|Price||479.16 $ Plus shipping costs $45.00|
|Availability||Will be delivered in 4 to 5 Business Days|
|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|
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 7.5150 mM NaCl0.5 mM EDTA0.5% NP401 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 7.5150 mM NaCl0.5 mM EDTA1 mM PMSF freshly added (optional)1x Protease Inhibitor Cocktail (e.g. Serva) freshly added. Wash-buffer 10 mM Tris/Cl pH 7.5150 - 500 mM NaCl0.5 mM EDTA1 mM PMSF freshly added (optional)1x Protease Inhibitor Cocktail (e.g. Serva®) freshly added. RIPA-Buffer (for cell lysis): 10 mM Tris/Cl, pH 7.5150 mM NaCl0.1% SDS1% TX1001% Deoxycholate5 mM EDTA1 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.|
|Restrictions||For Research Use only|
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