GFP-Trap® M (coupled to magnetic beads)

Application: Immunoprecipitation (IP), Mass Spectrometry (MS), Enzyme Activity Assay (EAA)

Catalog no.: ABIN1082214

Additional Information

Description: Green fluorescent proteins (GFP) and variants thereof are widely used to study proteinlocalization and dynamics. For biochemical analyses including mass spectroscopy and enzyme activity measurements these GFP-fusion proteins and their interacting factors can beisolated fast and efficiently (one step) via immunoprecipitation using the GFP-Trap®. Since theinteraction is mediated by a small GFP-binding protein coupled to magnetic particles the GFPTrap®_M enables purification of any protein of interest fused to GFP, eGFP, YFP or Venus.

Characteristics: particle size 0.5 - 1 µm

Comments:

For the immunoprecipitation of GFP-fusion proteins from cellular extracts.

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 freshly added (optinal)
1x mammalian Protease Inhibitor Cocktail (e.g. Serva®) freshly added
(optional for nuclear proteins / chromatin proteins:
DNaseI final conc. 1 myg/myl
2.5 mM MgCl2)
Dilution-buffer
10 mM Tris/Cl pH7.5
150 mM NaCl
0.5 mM EDTA
1 mM PMSF freshly added (options)
1x Protease Inhibitor Cocktail (e.g. Serva) freshly added
Wash-buffer
10 mM Tris/Cl pH7.5
150 mM NaCl
0.01% NP-40
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 pH7.5
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

Assay Procedure: 1. For one immunoprecipitation reaction resuspend cell pellet (~107 cells) in 50 - 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 pre-cooled cup. Adjust volume with dilution buffer to 250 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 10 - 50 µl cell lysate with 50 µl 4x SDS-sample buffer (-> refer as input)
5. Equilibrate GFP-Trap®_M in dilution buffer (if necessary). Resuspend magnetic particles and transfer calculated volume (20 - 30 µl) in a new reaction cup with 250 µl ice cold dilution buffer . Magnetically separate until supernatant is clear and wash twice with 250 µl of cold dilution buffer
(note: magnetic separation can be improved by adding 0.01% detergent (f.c.) to the dilution buffer)
6. Add cell lysate (200 – 1000 µl) to equilibrated GFP-Trap®_M
7. Incubate the GFP-Trap®_M 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®_M the final concentration of detergents should not exceed 0.2% to avoid unspecific binding to the matrix).
8. Magnetically separate until supernatant is clear
9. For western blot analysis dilute 50 µl supernatant with 50 µl 4x SDS-sample buffer (-> refer as non-bound). Discard remaining supernatant
10. Wash magnetic particles two times with 250 µl – 400 µl ice cold wash buffer
(optional: increase salt concentration in the second washing step up to 500 mM)
(note: magnetic separation can be improved by adding 0.01% detergent (f.c.) to the wash buffer )
11. Resuspend magnetic particle in 100 µl 2x SDS-Sample buffer GFP-Trap®_M manual - 2 - Version 2011-05-10
12. Boil resuspended beads for 10 minutes at 95°C to dissociate the immunocomplexes from the beads. Magnetically separate the GFP-Trap®_M and transfer supernatant to a fresh cup. SDS-PAGE should be performed with the supernatant.(-> refer as bound)
13. (optional) elute bound proteins by adding 50 µl 0.2 M glycine pH 2.5 (incubation time: 30 sec under constant mixing) followed by magnetic separation. 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.

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 antibodies Immunoprecipitations (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).

Gooding, Edge, Lorenz et al.: "MBNL1 and PTB cooperate to repress splicing of Tpm1 exon 3." in: Nucleic acids research, 2013 (PubMed).