GFP-Trap® A (coupled to Agarose beads)

Conjugate: Agarose Beads

Catalog no.: ABIN1082213

Additional Information

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

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

Comments:

For biochemical analyses including mass spectroscopy and enzyme activity measurements.

Application Details

Principle: for biochemical analysis of GFP fusion proteins and their interacting partners.

Reagent Preparation:
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 myg/myl2.5 mM MgCl2)
Dilution-buffer
10 mM Tris/Cl pH7.5
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
Wash-buffer
10 mM Tris/Cl pH7.5
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

Assay Procedure: 1. For one immunoprecipitation reaction resuspend cell pellet (~107 cells) in 200 µl lysisbuffer 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 pre-cooled tube. Adjust volume with dilution buffer to 500 µl –1000 myl. Discard pelletThe 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 in500 µl ice cold dilution buffer and spin down at 2700x g for 2 minutes at 4°C. Discardsupernatant 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 withthe 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 concentrationof 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 mylsupernatant with 50 µl 4x SDS-sample buffer (-> refer as non-bound), Discard remainingsupernatant
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 fromthe beads. The beads can be collected by centrifugation at 2700x g for 2 minutes at 4 °Cand 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: 30sec under constant mixing) followed by centrifugation. Transfer the supernatant to a freshcup and add 5 µl 1M Tris-base (pH 10.4) for neutralization. To increase the elutionefficiency this step can be repeated).

Concentration: 250 µl resin

Buffer: PBS

Preservative: 0.01% Sodium azide

Storage: Shipped at ambient temperature. Upon receipt store at 4°C. Do not freeze.

Restrictions: For Research Use only

Images

Comparison of GFP-Trap® with conventional mono- and polyclonal antibodiesImmunoprecipitations (IP) of GFP from protein extracts of GFP-producing human cells Input (I), non-bound (FT) and bound (B) fractions were separated by SDS-PAGE followed by Coomassie staining and Western Blotting. (hc) heavy chain, (lc) light chain of conventional antibodies.

Comparison of GFP-Trap_A and GFP-Trap_M 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

Product: Wild, Farhan, McEwan et al.: "Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth." in: Science (New York, N.Y.), Vol. 333, Issue 6039, pp. 228-33, 2011 (PubMed).

Majumdar, Cesario, White-Grindley et al.: "Critical role of amyloid-like oligomers of Drosophila Orb2 in the persistence of memory." in: Cell, Vol. 148, Issue 3, pp. 515-29, 2012 (PubMed).

Metzger, Gache, Xu et al.: "MAP and kinesin-dependent nuclear positioning is required for skeletal muscle function." in: Nature, Vol. 484, Issue 7392, pp. 120-4, 2012 (PubMed).

Gudesblat, Schneider-Pizoń, Betti et al.: "SPEECHLESS integrates brassinosteroid and stomata signalling pathways." in: Nature cell biology, Vol. 14, Issue 5, pp. 548-54, 2012 (PubMed).

Ries, Kaplan, Platonova et al.: "A simple, versatile method for GFP-based super-resolution microscopy via nanobodies." in: Nature methods, Vol. 9, Issue 6, pp. 582-4, 2012 (PubMed).

Pichler, Jack, Wolf et al.: "Versatile toolbox for high throughput biochemical and functional studies with fluorescent fusion proteins." in: PLoS ONE, Vol. 7, Issue 5, pp. e36967, 2012 (PubMed).

Castello, Fischer, Eichelbaum et al.: "Insights into RNA biology from an atlas of mammalian mRNA-binding proteins." in: Cell, Vol. 149, Issue 6, pp. 1393-406, 2012 (PubMed).