You are viewing an incomplete version of our website. Please click to reload the website as full version.

GFP-Trap® A

Details for Product No. ABIN1082213, Supplier: Log in to see
Antigen
Reactivity
Aequorea victoria
10
Host
Camelidae
Antibody Type
Recombinant Antibody
Conjugate
Agarose Beads
Application
Affinity Measurement (AM), Chromatin Immunoprecipitation (ChIP), Enzyme Activity Assay (EAA), Immunoprecipitation (IP), Mass Spectrometry (MS), Protein Complex Immunoprecipitation (Co-IP), Pull-Down Assay (Pull-Down), Purification (Purif)
Supplier
Log in to see
Supplier Product No.
Log in to see
Request

Get this product for free

It's quick and easy to submit your validation proposal. I want to validate this product

Learn more

Available images

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
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
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 ~ 90 µm

Assay Time 1.5 h
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®_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 2.5 mL resin
Buffer 20% EtOH
Handling Advice Do not freeze.
Storage 4 °C
Expiry Date 12 months
Supplier Images
 image for GFP-Trap® A (ABIN1082213) Left (IP): Pulldown of GFP with GFP-Trap®_A and GFP-Trap®_M from 293T cell extracts. ...
Western Blotting (WB) image for GFP-Trap® A (ABIN1082213) Comparison of GFP-Trap with conventional mono- and polyclonal antibodies: Immunopreci...
Product cited in: Braiterman, Gupta, Chaerkady, Cole, Hubbard: "Communication between the N- and C-termini is required for Cu-stimulated Ser/Thr phosphorylation of Cu-(I)ATPase (ATP7B)." in: The Journal of biological chemistry, 2015

Yan, Chu, Qin, Wang, Liu, Jin, Zhang, Gomez, Hergovich, Chen, He, Gao, Yao: "Regulation of NDR1 activity by PLK1 ensures proper spindle orientation in mitosis." in: Scientific reports, Vol. 5, pp. 10449, 2015

Xiao, MacNair, McGoldrick, McKeever, McLean, Zhang, Keith, Zinman, Rogaeva, Robertson: "Isoform Specific Antibodies Reveal Distinct Subcellular Localizations of C9orf72 in Amyotrophic Lateral Sclerosis." in: Annals of neurology, 2015

Satpathy, Wagner, Beli, Gupta, Kristiansen, Malinova, Francavilla, Tolar, Bishop, Hostager, Choudhary: "Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation." in: Molecular systems biology, Vol. 11, Issue 6, pp. 810, 2015

Hyodo, Taniguchi, Manabe, Kaido, Mise, Sugawara, Taniguchi, Okuno: "Phosphatidic acid produced by phospholipase D promotes RNA replication of a plant RNA virus." in: PLoS pathogens, Vol. 11, Issue 5, pp. e1004909, 2015

Vardabasso, Gaspar-Maia, Hasson, Pünzeler, Valle-Garcia, Straub, Keilhauer, Strub, Dong, Panda, Chung, Yao, Singh, Segura, Fontanals-Cirera, Verma, Mann, Hernando, Hake, Bernstein: "Histone Variant H2A.Z.2 Mediates Proliferation and Drug Sensitivity of Malignant Melanoma." in: Molecular cell, Vol. 59, Issue 1, pp. 75-88, 2015

Mochida, Oikawa, Kimura, Kirisako, Hirano, Ohsumi, Nakatogawa: "Receptor-mediated selective autophagy degrades the endoplasmic reticulum and the nucleus." in: Nature, Vol. 522, Issue 7556, pp. 359-62, 2015

Voit, Seiler, Grummt: "Cooperative Action of Cdk1/cyclin B and SIRT1 Is Required for Mitotic Repression of rRNA Synthesis." in: PLoS genetics, Vol. 11, Issue 5, pp. e1005246, 2015

Van Itallie, Tietgens, Krystofiak, Kachar, Anderson: "A complex of ZO-1 and the BAR-domain protein TOCA-1 regulates actin assembly at the tight junction." in: Molecular biology of the cell, Vol. 26, Issue 15, pp. 2769-87, 2015

Ojeda, Robles-Valero, Barreira, Bustelo: "The disease-linked Glu-26-Lys mutant version of Coronin 1A exhibits pleiotropic and pathway-specific signaling defects." in: Molecular biology of the cell, Vol. 26, Issue 16, pp. 2895-912, 2015

Catinot, Huang, Huang, Tseng, Chen, Gu, Lo, Wang, Chen, Zimmerli: "ETHYLENE RESPONSE FACTOR 96 positively regulates Arabidopsis resistance to necrotrophic pathogens by direct binding to GCC elements of jasmonate - and ethylene-responsive defence genes." in: Plant, cell & environment, 2015

Avila, Lee, Torii: "Co-Immunoprecipitation of Membrane-Bound Receptors." in: The Arabidopsis book / American Society of Plant Biologists, Vol. 13, pp. e0180, 2015

Maro, Gao, Olechwier, Hung, Liu, Özkan, Zhen, Shen: "MADD-4/Punctin and Neurexin Organize C. elegans GABAergic Postsynapses through Neuroligin." in: Neuron, Vol. 86, Issue 6, pp. 1420-32, 2015

Reyes, Ocolotobiche, Marmisollé, Robles Luna, Borniego, Bazzini, Asurmendi, García: "Citrus psorosis virus 24K protein interacts with citrus miRNA precursors, affects their processing and subsequent miRNA accumulation and target expression." in: Molecular plant pathology, 2015

Heinick, Husser, Himmler, Kirchhefer, Nunes, Schulte, Seidl, Rolfes, Dedman, Kaetzel, Gerke, Schmitz, Müller: "Annexin A4 is a novel direct regulator of adenylyl cyclase type 5." in: FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2015

Wang, Cordewener, America, Shan, Bouwmeester, Govers: "Arabidopsis lectin receptor kinases LecRK-IX.1 and LecRK-IX.2 are functional analogs in regulating Phytophthora resistance and plant cell death." in: Molecular plant-microbe interactions : MPMI, 2015

Sun, Li, Lu, Williams, Kao: "Pollen S-locus F-box proteins of Petunia involved in S-RNase-based self-incompatibility are themselves subject to ubiquitin-mediated degradation." in: The Plant journal : for cell and molecular biology, Vol. 83, Issue 2, pp. 213-223, 2015

Verheyen, Görnemann, Verbinnen, Boens, Beullens, Van Eynde, Bollen: "Genome-wide promoter binding profiling of protein phosphatase-1 and its major nuclear targeting subunits." in: Nucleic acids research, Vol. 43, Issue 12, pp. 5771-84, 2015

Yukawa, Ikebe, Toda: "The Msd1-Wdr8-Pkl1 complex anchors microtubule minus ends to fission yeast spindle pole bodies." in: The Journal of cell biology, Vol. 209, Issue 4, pp. 549-62, 2015

Frey, Lahmann, Hartmann, Seiler, Pöggeler: "Deletion of Smgpi1 encoding a GPI-anchored protein suppresses sterility of the STRIPAK mutant ΔSmmob3 in the filamentous ascomycete Sordaria macrospora." in: Molecular microbiology, Vol. 97, Issue 4, pp. 676-97, 2015