RFP-Trap® M

Details for Product No. ABIN1082218
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Target Name (Antigen)
Synonyms RFP, RNF76
Reactivity
Discosoma
(12)
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 5 references available
Quantity 10 tests
Options
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Request Want additional data for this product?

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

Catalog No. ABIN1082218
221.10 $
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Purpose RFP-Trap® is a high quality RFP-binding protein coupled to a monovalent matrix (magnetic particles) 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®_M slurry binds 0.25 – 0.5 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 magnetic particles
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 0.5 - 1 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®_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 RFP-Trap®_M beads and incubate the RFPTrap®_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 RFP-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 RFP-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 250 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
RFP-Trap® M 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: 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).

Validation Images
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