RFP-Trap® A

Details for Product No. ABIN1082217
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Target Name (Antigen)
Synonyms RFP, RNF76
Reactivity
Discosoma
(12)
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 26 references available
Quantity 10 tests
Options
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Request Want additional data for this product?

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Catalog No. ABIN1082217
221.10 $
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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
Fragment heavy chain antibody (hcAb)
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
Alternative Name RFP
Research Area Tags/Labels
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®. Since the interaction is mediated by a small RFP binding protein coupled to agarose beads the RFPTrap®_M enables purification of any protein of interest fused to RFP (monomeric derivates of DsRed, including mRFP1, mCherry, mPlum, mOrange).
Comment

Bead size ~ 90 µm

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 250 µl resin
Buffer 20% EtOH
Handling Advice Do not freeze.
Storage 4 °C
Expiry Date 12 months
Supplier Images
RFP-Trap® A RFP-Trap® to immunoprecipitate red fluorescent proteins Pulldown of different monomeric red fluorescent proteins mRFP, mCherry and mOrange from cell extracts of human cells. Input (I), non-bound (FT) and bound (B) fractions were separated by SDS-PAGE followed by Coomassie staining and Western Blotting.
Product cited in: Pollithy, Romer, Lang et al.: "Magnetosome expression of functional camelid antibody fragments (nanobodies) in Magnetospirillum gryphiswaldense." in: Applied and environmental microbiology, Vol. 77, Issue 17, pp. 6165-71, 2011 (PubMed).

Aboobakar, Wang, Heitman et al.: "The C2 domain protein Cts1 functions in the calcineurin signaling circuit during high-temperature stress responses in Cryptococcus neoformans." in: Eukaryotic cell, Vol. 10, Issue 12, pp. 1714-23, 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).

Moutin, Raynaud, Fagni et al.: "GKAP-DLC2 interaction organizes the postsynaptic scaffold complex to enhance synaptic NMDA receptor activity." in: Journal of cell science, Vol. 125, Issue Pt 8, pp. 2030-40, 2012 (PubMed).

Lefebvre, Klaus-Heisen, Pietraszewska-Bogiel et al.: "Role of N-glycosylation sites and CXC motifs in trafficking of medicago truncatula Nod factor perception protein to plasma membrane." in: The Journal of biological chemistry, Vol. 287, Issue 14, pp. 10812-23, 2012 (PubMed).

Zanet, Jayo, Plaza et al.: "Fascin promotes filopodia formation independent of its role in actin bundling." in: The Journal of cell biology, Vol. 197, Issue 4, pp. 477-86, 2012 (PubMed).

Peña, Robles Luna, Zanek et al.: "Citrus psorosis and Mirafiori lettuce big-vein ophiovirus coat proteins localize to the cytoplasm and self interact in vivo." in: Virus research, Vol. 170, Issue 1-2, pp. 34-43, 2012 (PubMed).

Ulbricht, Alzrigat, Horch et al.: "PML promotes MHC class II gene expression by stabilizing the class II transactivator." in: The Journal of cell biology, Vol. 199, Issue 1, pp. 49-63, 2012 (PubMed).

Streitner, Köster, Simpson et al.: "An hnRNP-like RNA-binding protein affects alternative splicing by in vivo interaction with transcripts in Arabidopsis thaliana." in: Nucleic acids research, Vol. 40, Issue 22, pp. 11240-55, 2012 (PubMed).

Abdallah, Zhou, Kim et al.: "Activated Cdc42 kinase regulates Dock localization in male germ cells during Drosophila spermatogenesis." in: Developmental biology, 2013 (PubMed).

Robles Luna, Peña, Borniego et al.: "Ophioviruses CPsV and MiLBVV movement protein is encoded in RNA 2 and interacts with the coat protein." in: Virology, Vol. 441, Issue 2, pp. 152-61, 2013 (PubMed).

Ono, Yamada, Endo et al.: "Analysis of human protein replacement stable cell lines established using snoMEN-PR vector." in: PLoS ONE, Vol. 8, Issue 4, pp. e62305, 2013 (PubMed).

Montesinos, Langhans, Sturm et al.: "Putative p24 complexes in Arabidopsis contain members of the delta and beta subfamilies and cycle in the early secretory pathway." in: Journal of experimental botany, Vol. 64, Issue 11, pp. 3147-67, 2013 (PubMed).

Ramanujam, Calvert, Selvaraj et al.: "The late endosomal HOPS complex anchors active G-protein signaling essential for pathogenesis in magnaporthe oryzae." in: PLoS pathogens, Vol. 9, Issue 8, pp. e1003527, 2013 (PubMed).

Sun, Li, Suo et al.: "Global analysis of fission yeast mating genes reveals new autophagy factors." in: PLoS genetics, Vol. 9, Issue 8, pp. e1003715, 2013 (PubMed).

Petrovská, Jerábková, Kohoutová et al.: "Overexpressed TPX2 causes ectopic formation of microtubular arrays in the nuclei of acentrosomal plant cells." in: Journal of experimental botany, Vol. 64, Issue 14, pp. 4575-87, 2013 (PubMed).

Bauch, Koliwer, Buck et al.: "Subcellular sorting of the G-protein coupled mouse somatostatin receptor 5 by a network of PDZ-domain containing proteins." in: PLoS ONE, Vol. 9, Issue 2, pp. e88529, 2014 (PubMed).

Hagen, Mattay, Räuber et al.: "Characterization and inhibition of AF10-mediated interaction." in: Journal of peptide science : an official publication of the European Peptide Society, Vol. 20, Issue 6, pp. 385-97, 2014 (PubMed).

Mass, Wachten, Aschenbrenner et al.: "Murine Creld1 controls cardiac development through activation of calcineurin/NFATc1 signaling." in: Developmental cell, Vol. 28, Issue 6, pp. 711-26, 2014 (PubMed).

Janbon, Ormerod, Paulet et al.: "Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation. ..." in: PLoS genetics, Vol. 10, Issue 4, pp. e1004261, 2014 (PubMed).

Yepes, Koch, Waldvogel et al.: "Reconstruction of mreB expression in Staphylococcus aureus via a collection of new integrative plasmids." in: Applied and environmental microbiology, Vol. 80, Issue 13, pp. 3868-78, 2014 (PubMed).

Sechi, Colotti, Belloni et al.: "GOLPH3 is essential for contractile ring formation and Rab11 localization to the cleavage site during cytokinesis in Drosophila melanogaster." in: PLoS genetics, Vol. 10, Issue 5, pp. e1004305, 2014 (PubMed).

Jaiswal, Lauritzen, Scheffer et al.: "S100A11 is required for efficient plasma membrane repair and survival of invasive cancer cells." in: Nature communications, Vol. 5, pp. 3795, 2014 (PubMed).

Niehl, Pasquier, Ferriol et al.: "Comparison of the Oilseed rape mosaic virus and Tobacco mosaic virus movement proteins (MP) reveals common and dissimilar MP functions for tobamovirus spread." in: Virology, Vol. 456-457, pp. 43-54, 2014 (PubMed).

Stagi, Klein, Gould et al.: "Lysosome size, motility and stress response regulated by fronto-temporal dementia modifier TMEM106B." in: Molecular and cellular neurosciences, Vol. 61, pp. 226-40, 2014 (PubMed).

Köster, Meyer, Weinholdt et al.: "Regulation of pri-miRNA processing by the hnRNP-like protein AtGRP7 in Arabidopsis." in: Nucleic acids research, Vol. 42, Issue 15, pp. 9925-36, 2014 (PubMed).

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