GFP-Trap® M (coupled to magnetic particles) Kit

Antigen: Green Fluorescent Protein (GFP)

Applikation: Immunpräzipitation (IP)

Produktnummer: ABIN509406

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: magnetic GFP-Trap®-M (size ~ 0.5 -1 μM in PBS. 0.1% BSA, 0.01% NaN3 )

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.