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RFP-Trap® Agarose Kit

AM, ChIP, EAA, IP, MS, Co-IP, Pull-Down, Purif Reactivity: Discosoma Cell Extracts
Pubmed (55)
Catalog No. ABIN1082217
$330.00
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  • Target
    RFP
    Antibody Type
    Recombinant Antibody
    Fragment
    single-domain Antibody (sdAb)
    Reactivity
    Discosoma
    Host
    • 5
    Camelid (Camelidae)
    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)
    Purpose
    RFP-Trap® is a high quality RFP-binding protein coupled to a monovalent matrix (agarose beads) for biochemical analysis of RFP fusion proteins and their interacting partners.
    Brand
    RFP-Trap®
    Sample Type
    Cell Extracts
    Specificity
    Binding capacity: 10 µl RFP-Trap®_A slurry binds 2.5 - 3 µg of RFP
    Cross-Reactivity (Details)
    RFP-Trap efficiently pulls down various Red fluorescent proteins derived from DsRed, e.g. mRFP1, mCherry, mOrange, mPlum but also mRuby, mKate2 and RFP-tagged fusion proteins. No cross-reaction to DsRed, mRFPruby, TagRFP and all GFPs 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
    RFP-Trap® coupled to agarose beads
    Material not included
    Lysis buffer (CoIP), 10x RIPA buffer, Dilution buffer, Wash buffer, Elution buffer
  • Application Notes
    Red fluorescent proteins (RFP) and variants thereof are widely used to study protein localization and dynamics. For biochemical analyses including mass spectroscopy and enzyme activity measurements these RFP-fusion proteins and their interacting factors can be isolated fast and efficiently (one step) via immunoprecipitation using the RFP-Trap. The RFPTrap_A enables purification of any protein of interest fused to RFP.
    Comment

    Bead size ~ 90 µm

    Assay Time
    1.5 h
    Protocol
    • Robust and versatile tool for biochemical analyses of RFP-fusion proteins
    • Short incubation times (5 - 30 min)
    • Quantitative isolation of fusion proteins and transiently bound factors from cell extracts or organelles
    • Low unspecifi c 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 RFP-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 RFP-Trap®_A beads and incubate the RFP-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 RFP-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 RFP-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
  • Bieluszewska, Weglewska, Bieluszewski, Lesniewicz, Poreba: "PKA-binding domain of AKAP8 is essential for direct interaction with DPY30 protein." in: The FEBS journal, Vol. 285, Issue 5, pp. 947-964, 2019 (PubMed).

    Zhang, Liang, Naqvi, Lin, Qian, Zhang, Deng: "Phototrophy and starvation-based induction of autophagy upon removal of Gcn5-catalyzed acetylation of Atg7 in Magnaporthe oryzae." in: Autophagy, Vol. 13, Issue 8, pp. 1318-1330, 2018 (PubMed).

    Meyer, Köster, Nolte, Weinholdt, Lewinski, Grosse, Staiger: "Adaptation of iCLIP to plants determines the binding landscape of the clock-regulated RNA-binding protein AtGRP7." in: Genome biology, Vol. 18, Issue 1, pp. 204, 2018 (PubMed).

    Willett, Martina, Zewe, Wills, Hammond, Puertollano: "TFEB regulates lysosomal positioning by modulating TMEM55B expression and JIP4 recruitment to lysosomes." in: Nature communications, Vol. 8, Issue 1, pp. 1580, 2018 (PubMed).

    Ramat, Hannaford, Januschke: "Maintenance of Miranda Localization in Drosophila Neuroblasts Involves Interaction with the Cognate mRNA." in: Current biology : CB, Vol. 27, Issue 14, pp. 2101-2111.e5, 2018 (PubMed).

    MacLennan, García-Cañadas, Reichmann, Khazina, Wagner, Playfoot, Salvador-Palomeque, Mann, Peressini, Sanchez, Dobie, Read, Hung, Eskeland, Meehan, Weichenrieder, García-Pérez, Adams: "Mobilization of LINE-1 retrotransposons is restricted by Tex19.1 in mouse embryonic stem cells." in: eLife, Vol. 6, 2018 (PubMed).

    Thillaiappan, Chavda, Tovey, Prole, Taylor: "Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions." in: Nature communications, Vol. 8, Issue 1, pp. 1505, 2018 (PubMed).

    Seung, Boudet, Monroe, Schreier, David, Abt, Lu, Zanella, Zeeman: "Homologs of PROTEIN TARGETING TO STARCH Control Starch Granule Initiation in Arabidopsis Leaves." in: The Plant cell, Vol. 29, Issue 7, pp. 1657-1677, 2018 (PubMed).

    Kulkarni, Tan, Syed Sulaiman, Lamar, Bansal, Cui, Qiao, Ito: "RUNX1 and RUNX3 protect against YAP-mediated EMT, stem-ness and shorter survival outcomes in breast cancer." in: Oncotarget, Vol. 9, Issue 18, pp. 14175-14192, 2018 (PubMed).

    Thompson, Morrison, Shirran, Groen, Gillingwater, Botting, Sleeman: "Neurochondrin interacts with the SMN protein suggesting a novel mechanism for spinal muscular atrophy pathology." in: Journal of cell science, Vol. 131, Issue 8, 2018 (PubMed).

    Jiang, Wei, Long, Owen, Wang, Wu, Luo, Dang, Ma: "A genetic program mediates cold-warming response and promotes stress-induced phenoptosis in C. elegans." in: eLife, Vol. 7, 2018 (PubMed).

    Li, Luo, Wang, Liu, Chen, Zhao, Tan, Wang, Qin, Li, Xu, Yang: "The REN4 rheostat dynamically coordinates the apical and lateral domains of Arabidopsis pollen tubes." in: Nature communications, Vol. 9, Issue 1, pp. 2573, 2018 (PubMed).

    Krapp, Schuy, Greiner, Stephan, Alberter, Funk, Marschall, Wege, Bailer, Kleinow, Krenz: "Begomoviral Movement Protein Effects in Human and Plant Cells: Towards New Potential Interaction Partners." in: Viruses, Vol. 9, Issue 11, 2018 (PubMed).

    Harlen, Churchman: "Subgenic Pol II interactomes identify region-specific transcription elongation regulators." in: Molecular systems biology, Vol. 13, Issue 1, pp. 900, 2017 (PubMed).

    Sechi, Frappaolo, Fraschini, Capalbo, Gottardo, Belloni, Glover, Wainman, Giansanti: "Rab1 interacts with GOLPH3 and controls Golgi structure and contractile ring constriction during cytokinesis in Drosophila melanogaster." in: Open biology, Vol. 7, Issue 1, 2017 (PubMed).

    Shwab, Juvvadi, Waitt, Soderblom, Moseley, Nicely, Asfaw, Steinbach: "A Novel Phosphoregulatory Switch Controls the Activity and Function of the Major Catalytic Subunit of Protein Kinase A in Aspergillus fumigatus." in: mBio, Vol. 8, Issue 1, 2017 (PubMed).

    Homsi, Lang: "The specificity of homomeric clustering of CD81 is mediated by its δ-loop." in: FEBS open bio, Vol. 7, Issue 2, pp. 274-283, 2017 (PubMed).

    Parhad, Tu, Weng, Theurkauf: "Adaptive Evolution Leads to Cross-Species Incompatibility in the piRNA Transposon Silencing Machinery." in: Developmental cell, Vol. 43, Issue 1, pp. 60-70.e5, 2017 (PubMed).

    Diamanti, Gupta, Bennecke, De Oliveira, Ramakrishnan, Braczynski, Richter, Beli, Hu, Saleh, Mittelbronn, Dikic, Greten: "IKKα controls ATG16L1 degradation to prevent ER stress during inflammation." in: The Journal of experimental medicine, Vol. 214, Issue 2, pp. 423-437, 2017 (PubMed).

    Kumar, Cheok: "Dynamics of RIF1 SUMOylation is regulated by PIAS4 in the maintenance of Genomic Stability." in: Scientific reports, Vol. 7, Issue 1, pp. 17367, 2017 (PubMed).

  • Target
    RFP
    Alternative Name
    RFP
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