GFP-Trap® M

Details for Product No. ABIN509401
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Target Name (Antigen)
Reactivity
Aequorea victoria
(21)
Host
Camelidae
Conjugate
Magnetic Particles
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 21 references available
Quantity 20 tests
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Catalog No. ABIN509401
418.75 $
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Purpose GFP-Trap® is a high quality GFP-binding protein coupled to a monovalent matrix (magnetic particles) 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®_M slurry binds 0.25 – 0.5 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 magnetic particles
Material not included Lysis buffer (CoIP), 10x RIPA buffer, Dilution buffer, Wash buffer, Elution buffer
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 0.5 - 1 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)
Reagent Preparation Suggested buffer composition

  • Lysis buffer (CoIP): 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA,0.5% NP-40
  • 10x RIPA buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% Deoxycholate
  • Dilution buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA
  • Wash buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA
  • Elution buffer: 200 mM glycine pH 2.5
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®_M beads in dilution buffer. Resuspend magnetic beads by vortexing and transfer 20 - 30 µL bead slurry in 500 µL ice cold dilution buffer. Magnetically separate beads until supernatant is clear. Discard supernatant and wash beads 2 more times with 500 µL ice cold dilution buffer.
  • 6. Add cell lysate to equilibrated GFP-Trap®_M beads and incubate the GFPTrap®_M 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®_M the final concentration of detergents should not exceed 0.2% to avoid unspecific binding to the matrix.
  • 7. Magnetically separate beads until supernatant is clear. For western blot analysis dilute 50 µL supernatant with 50 µL 2x SDS-sample buffer (à refer to as nonbound). 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®_M 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 magnetically separated 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 1 x PBS,0.01% Sodium azide
Preservative Sodium azide
Precaution of Use This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Handling Advice Do not freeze.
Storage 4 °C
Expiry Date 12 months
Supplier Images
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.
Product cited in: Lahiri, Chao, Tavassoli et al.: "A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria." in: PLoS biology, Vol. 12, Issue 10, pp. e1001969, 2014 (PubMed).

Montesinos, Pastor-Cantizano, Robinson et al.: "Arabidopsis p24δ5 and p24δ9 facilitate COPI-dependent Golgi-to-ER transport of the K/HDEL receptor ERD2." in: The Plant journal : for cell and molecular biology, 2014 (PubMed).

Cruz-García, López-Saavedra, Huertas: "BRCA1 Accelerates CtIP-Mediated DNA-End Resection." in: Cell reports, Vol. 9, Issue 2, pp. 451-9, 2014 (PubMed).

Webster, Colombi, Jäger et al.: "Surveillance of Nuclear Pore Complex Assembly by ESCRT-III/Vps4." in: Cell, Vol. 159, Issue 2, pp. 388-401, 2014 (PubMed).

Chen, Yeap, Bogoyevitch: "The JNK1/JNK3 interactome - Contributions by the JNK3 unique N-terminus and JNK common docking site residues." in: Biochemical and biophysical research communications, 2014 (PubMed).

Patten, Wong, Khacho et al.: "OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand." in: The EMBO journal, 2014 (PubMed).

Shen, Ding, Gao et al.: "N-linked glycosylation of AtVSR1 is important for vacuolar protein sorting in Arabidopsis." in: The Plant journal : for cell and molecular biology, 2014 (PubMed).

Tanaka, Tan, Mochida et al.: "Hrr25 triggers selective autophagy-related pathways by phosphorylating receptor proteins." in: The Journal of cell biology, Vol. 207, Issue 1, pp. 91-105, 2014 (PubMed).

Shahid, Soroka, Kong et al.: "Structure and mechanism of action of the BRCA2 breast cancer tumor suppressor." in: Nature structural & molecular biology, Vol. 21, Issue 11, pp. 962-8, 2014 (PubMed).

De Wever, Nasa, Chamousset et al.: "The human mitotic kinesin KIF18A binds protein phosphatase 1 (PP1) through a highly conserved docking motif." in: Biochemical and biophysical research communications, 2014 (PubMed).

McGough, Steinberg, Gallon et al.: "Identification of molecular heterogeneity in SNX27-retromer-mediated endosome-to-plasma membrane recycling." in: Journal of cell science, 2014 (PubMed).

Falcone, Roman, Hnia et al.: "N-WASP is required for Amphiphysin-2/BIN1-dependent nuclear positioning and triad organization in skeletal muscle and is involved in the pathophysiology of centronuclear myopathy." in: EMBO molecular medicine, Vol. 6, Issue 11, pp. 1455-75, 2014 (PubMed).

Cobret, De Tauzia, Ferent et al.: "Targeting the Cis-Dimerization of Lingo-1 with Small-Molecule Affects Its Downstream Signaling." in: British journal of pharmacology, 2014 (PubMed).

van der Lelij, Stocsits, Ladurner et al.: "SNW1 enables sister chromatid cohesion by mediating the splicing of sororin and APC2 pre-mRNAs." in: The EMBO journal, 2014 (PubMed).

Yatsenko, Marrone, Shcherbata: "miRNA-based buffering of the cobblestone-lissencephaly-associated extracellular matrix receptor dystroglycan via its alternative 3'-UTR." in: Nature communications, Vol. 5, pp. 4906, 2014 (PubMed).

Fouquerel, Goellner, Yu et al.: "ARTD1/PARP1 negatively regulates glycolysis by inhibiting hexokinase 1 independent of NAD+ depletion." in: Cell reports, Vol. 8, Issue 6, pp. 1819-31, 2014 (PubMed).

Chan, West: "Spatial control of the GEN1 Holliday junction resolvase ensures genome stability." in: Nature communications, Vol. 5, pp. 4844, 2014 (PubMed).

Asante, Stevenson, Stephens: "Subunit composition of the human cytoplasmic dynein-2 complex." in: Journal of cell science, 2014 (PubMed).

Zhang, Liu, Ding et al.: "Fission yeast Pxd1 promotes proper DNA repair by activating Rad16XPF and inhibiting Dna2." in: PLoS biology, Vol. 12, Issue 9, pp. e1001946, 2014 (PubMed).

Blasius, Wagner, Choudhary et al.: "A quantitative 14-3-3 interaction screen connects the nuclear exosome targeting complex to the DNA damage response." in: Genes & development, Vol. 28, Issue 18, pp. 1977-82, 2014 (PubMed).

Gauthier-Kemper, Igaev, Sündermann et al.: "Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43." in: Molecular biology of the cell, Vol. 25, Issue 21, pp. 3284-99, 2014 (PubMed).

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