GFP-Trap®_A for Immunoprecipitation of GFP-Fusion Proteins
For the immunoprecipitation of GFP-fusion-proteins from cellular extracts.
Only for research applications, not for diagnostic or therapeutic use
1. Introduction
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®. Since the
interaction is mediated by a small GFP-binding protein coupled to agarose beads the GFP-
黄昏到寺蝙蝠飞Trap®_A enables purification of any protein of interest fused to GFP, eGFP, YFP or Venus. 2. Content
GFP-Trap®_A (bead size: ~ 80 µM) in 20% EtOH
Catalog no.: gta-20; 0.5 ml; 20 reactions
binding capacity: 10 µl GFP-Trap®_A slurry binds 2.5 – 3 µg of GFP (data obtained from
HEK293T cells expressing GFP only).
3. Stability and
Storage
Store material at 2 - 8°C, do not freeze. expiration time: 6 months after opening
4. 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
三棵树苏童
3. Spin cell lysate at 20.000x g for 5 -10 minutes at 4 °C
4. Transfer supernatant to a pre-cooled 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 -80°C.
For immunoblot analysis dilute 50 µl cell lysate with 50 µl 4x SDS-sample buffer
(-> refer as input)
5. Equilibrate GFP-Trap®_A 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 beads two more times with 500 µl ice cold dilution buffer.
6. Add cell lysate to equilibrated GFP-Trap®_A beads and incubate the GFP-Trap®_A with
the cell lysate under constant mixing for 10 min – 2 h at room temperature or 4°C
(note: during incubation of protein sample with the GFP-Trap®_A the final concentration
rcctof detergents should not exceed 0.2% to avoid unspecific binding to the matrix
7. Spin tube at 2700x 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
8. Wash beads pellet two times with 500 µl ice cold wash buffer
(optional: increase salt concentration in the second washing step up to 500 mM)
9. Resuspend GFP-Trap®_A beads in 100 µl 2x SDS-Sample buffer
10. 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)
11. (optional) elute bound proteins by adding 50 µl 0.2 M glycine pH 2.5 (incubation time: 30
sec under constant mixing) followed by centrifugation. Transfer the supernatant to a fresh
cup and add 5 µl 1M Tris-base (pH 10.4) for neutralization. To increase the elution
efficiency this step can be repeated)
Suggested Buffers (as tested in our laboratory)
Lysis-buffer (for CoIP):
10 mM Tris/Cl pH7.5
150 mM NaCl
0.5 mM EDTA
0.5% NP40
1 mM PMSF has to be freshly added (optional)
1x Protease Inhibitor Cocktail (e.g. Serva®) has to be freshly added (optional for nuclear proteins / chromatin proteins:
DNaseI final conc. 1 µg/µl
2.5 mM MgCl2)
Dilution-buffer
10 mM Tris/Cl pH7.5
150 mM NaCl
0.5 mM EDTA
11ccmm1 mM PMSF has to be freshly added (optional)
1x Protease Inhibitor Cocktail (e.g. Serva®) has to be freshly added Wash-buffer
150 mM NaCl
0.5 mM EDTA
1 mM PMSF has to be freshly added (optional)
1x Protease Inhibitor Cocktail (e.g. Serva®) has to be freshly added RIPA-Buffer (for cell lysis):
10 mM Tris/Cl pH7.5
150 mM NaCl
0.1% SDS
1% TX100
1% Deoxycholate
5 mM EDTA
1 mM PMSF has to be freshly added (optional)
1x Protease Inhibitor Cocktail (e.g. Serva®) has to be freshly added
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