GFP-Trap® A Kit

Details for Product No. ABIN509405
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Target Name (Antigen)
Reactivity
Aequorea victoria
(21)
Host
Camelidae
Conjugate
Agarose Beads
Application
Protein Complex Immunoprecipitation (Co-IP), Mass Spectrometry (MS), Enzyme Activity Assay (EAA), Affinity Measurement (AM), Chromatin Immunoprecipitation (ChIP), Pull-Down Assay (Pull-Down), Purification (Purif), Immunoprecipitation (IP)
Pubmed 19 references available
Catalog no. ABIN509405
Quantity 20 tests
Price
554.40 $   Plus shipping costs $45.00
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  • +1 404 474 4654
  • +1 888 205 9894 (TF)
Purpose GFP-Trap® is a high quality GFP-binding protein coupled to a monovalent matrix (agarose beads) for biochemical analysis of GFP fusion proteins and their interacting partners.
Brand GFP-Trap®
Sample Type Cell Extracts
Fragment heavy chain antibody (hcAb)
Specificity Binding capacity: 10 µL GFP-Trap®_A slurry binds 2.5 – 3 µg of GFP
Cross-Reactivity (Details) GFP-Trap® specifically binds to eGFP, wtGFP, GFP S65T, TagGFP, eYFP, YFP, Venus, Citrin, CFP. No binding to proteins derived from DsRed, all RFPs and TurboGFP can be detected.
Characteristics Antibodies – extremely powerful tools in biomedical research – are large complex molecules (~ 150 kDa) consisting of two heavy and two light chains. Due to their complex structure, the use of antibodies is often limited and hindered by batch-to-batch variations.

Camelidae (camels, dromedaries, llamas and alpacas) possess functional antibodies devoid of light chains, so-called heavy chain antibodies (hcAbs). hcAbs recognize and bind their antigens via a single variable domain (VHH). These VHH domains are the smallest intact antigen binding fragments (~ 13 kDa).

Nano-Traps are based on single domain antibody fragments (VHHs) derived from alpaca.
Components GFP-Trap® coupled to agarose beads
Lysis buffer (CoIP) 40 mL
10x RIPA buffer 2 mL
Dilution buffer 150 mL
Wash buffer 40 mL
Elution buffer 3 mL
Alternative Name GFP
Background The green fluorescent protein (GFP) and variants thereof are widely used to study the subcellular localization and dynamics of proteins. GFP fusion proteins can be expressed in different cell types at different expression levels by transient or stable transfection. Transient expression may provide quick informative results, however, in many cases it is necessary to generate stable cell lines that express the GFP fusion protein of interest at a level similar to the one of the endogenous protein. Quantification of GFP fusion proteins in cells can be tricky since existing methods, like fluorescence microscopy or Western Blotting, are often shows insufficient signal to noise ratios or high signal variabilities .
Research Area Tags/Labels
Application Notes 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.
Comment

Bead size ~ 90 µm

Protocol - Robust and versatile tool for biochemical analyses of GFP-fusion proteins
- Short incubation times (5 – 30 min)
- Quantitative isolation of fusion proteins and transiently bound factors from cell extracts or organelles
- Low unspecific binding
- No contaminating heavy and light chains of conventional antibodies
- Applicable in Chromatin Immunoprecipitation (ChIP)
Assay Procedure Before you start: Add 1ml PBS to your cells and scrape them off the petri dish.Transfer to precooled tube, spin 3 min at 500 x g and discard supernatant. Wash cell pellet twice with ice cold PBS, briefly resuspending the cells.
1. For one immunoprecipitation reaction resuspend cell pellet (~10^7 mammalian cells) in 200 µL lysis buffer by pipetting (or using a syringe).
optional: add 1 mM PMSF and Protease inhibitor cocktail (not included) to lysis buffer
optional for nuclear/chromatin proteins: add 1 mg/ml DNase and 2.5 mM MgCl2 (not included) to lysis buffer
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 µL. Discard pellet.
optional: add 1 mM PMSF and Protease inhibitor cocktail (not included) to dilution buffer
note: 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 2x SDS-sample buffer(à refer to as input).
5. Equilibrate GFP-Trap®_A beads in dilution buffer. Resuspend 20 - 30 µL bead slurry in 500 µL ice cold dilution buffer and spin down at 2.500x g for 2 minutes at 4°C. Discard supernatant and wash beads 2 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 beads 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 of detergents should not exceed 0.2% to avoid unspecific binding to the matrix
7. Spin tube at 2.500x g for 2 minutes at 4°C. For western blot analysis dilute 50 µL supernatant with 50 µL 2x SDS-sample buffer (à refer to as non-bound). Discard remaining supernatant.
8. Wash beads three times with 500 µL ice cold wash buffer. After the last wash step, transfer beads to new tube.
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 or go to step 11.
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 2.500x g for 2 minutes at 4°C and SDS-PAGE is performed with the supernatant (à refer to 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 elution efficiency this step can be repeated.
Restrictions For Research Use only
Concentration 500 µL resin
Buffer 20% EtOH
Handling Advice Do not freeze.
Storage 4 °C
Expiry Date 12 months
Supplier Images
GFP-Trap® A Kit 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.
Product cited in: Miroci, Schob, Kindler et al.: "Makorin Ring Zinc Finger Protein 1 (MKRN1), a Novel Poly(A)-binding Protein-interacting Protein, Stimulates Translation in Nerve Cells." in: The Journal of biological chemistry, Vol. 287, Issue 2, pp. 1322-34, 2012 (PubMed).

Jost, Rottach, Milden et al.: "Generation and characterization of rat and mouse monoclonal antibodies specific for MeCP2 and their use in X-inactivation studies." in: PLoS ONE, Vol. 6, Issue 11, pp. e26499, 2011 (PubMed).

Neumüller, Wirtz-Peitz, Lee et al.: "Stringent analysis of gene function and protein-protein interactions using fluorescently tagged genes." in: Genetics, Vol. 190, Issue 3, pp. 931-40, 2012 (PubMed).

von Maltzahn, Bentzinger, Rudnicki: "Wnt7a-Fzd7 signalling directly activates the Akt/mTOR anabolic growth pathway in skeletal muscle." in: Nature cell biology, Vol. 14, Issue 2, pp. 186-91, 2012 (PubMed).

Corrotte, Fernandes, Tam et al.: "Toxin Pores Endocytosed During Plasma Membrane Repair Traffic into the Lumen of MVBs for Degradation." in: Traffic (Copenhagen, Denmark), 2012 (PubMed).

Howitt, Lackovic, Low et al.: "Ndfip1 regulates nuclear Pten import in vivo to promote neuronal survival following cerebral ischemia." in: The Journal of cell biology, Vol. 196, Issue 1, pp. 29-36, 2012 (PubMed).

OLoghlen, Muñoz-Cabello, Gaspar-Maia et al.: "MicroRNA regulation of Cbx7 mediates a switch of Polycomb orthologs during ESC differentiation." in: Cell stem cell, Vol. 10, Issue 1, pp. 33-46, 2012 (PubMed).

Weissbach, Scadden: "Tudor-SN and ADAR1 are components of cytoplasmic stress granules." in: RNA (New York, N.Y.), 2012 (PubMed).

Zwaenepoel, Naba, da Cunha et al.: "Ezrin regulates microvillus morphogenesis by promoting distinct activities of Eps8 proteins." in: Molecular biology of the cell, 2012 (PubMed).

Magyar, Horváth, Khan et al.: "Arabidopsis E2FA stimulates proliferation and endocycle separately through RBR-bound and RBR-free complexes." in: The EMBO journal, 2012 (PubMed).

Borrego-Pinto, Jegou, Osorio et al.: "Samp1 is a component of TAN lines and is required for nuclear movement." in: Journal of cell science, Vol. 125, Issue Pt 5, pp. 1099-105, 2012 (PubMed).

Sala-Valdés, Gordón-Alonso, Tejera et al.: "Association of syntenin-1 with M-RIP polarizes Rac-1 activation during chemotaxis and immune interactions." in: Journal of cell science, Vol. 125, Issue Pt 5, pp. 1235-46, 2012 (PubMed).

Tibarewal, Zilidis, Spinelli et al.: "PTEN Protein Phosphatase Activity Correlates with Control of Gene Expression and Invasion, a Tumor-Suppressing Phenotype, But Not with AKT Activity." in: Science signaling, Vol. 5, Issue 213, pp. ra18, 2012 (PubMed).

Guttery, Ferguson, Poulin et al.: "A Putative Homologue of CDC20/CDH1 in the Malaria Parasite Is Essential for Male Gamete Development." in: PLoS pathogens, Vol. 8, Issue 2, pp. e1002554, 2012 (PubMed). Further details: GFP-tagged CDC20 proteins were then immunoprecipitated using GFP-TRAP beads

Beli, Lukashchuk, Wagner et al.: "Proteomic Investigations Reveal a Role for RNA Processing Factor THRAP3 in the DNA Damage Response." in: Molecular cell, 2012 (PubMed).

General Rothbauer, Zolghadr, Tillib et al.: "Targeting and tracing antigens in live cells with fluorescent nanobodies." in: Nature methods, Vol. 3, Issue 11, pp. 887-9, 2006 (PubMed).

Agarwal, Hardt, Brero et al.: "MeCP2 interacts with HP1 and modulates its heterochromatin association during myogenic differentiation." in: Nucleic acids research, Vol. 35, Issue 16, pp. 5402-8, 2007 (PubMed).

Rothbauer, Zolghadr, Muyldermans et al.: "A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins." in: Molecular & cellular proteomics : MCP, Vol. 7, Issue 2, pp. 282-9, 2008 (PubMed).

Trinkle-Mulcahy, Boulon, Lam et al.: "Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes." in: The Journal of cell biology, Vol. 183, Issue 2, pp. 223-39, 2008 (PubMed).

Reactivities (21)
Request Want additional data for this product?

The Independent Validation Initiative strives to provide you with high quality data. Find out more

Order hotline:

  • +1 404 474 4654
  • +1 888 205 9894 (TF)
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