RFP-Trap® Magnetic Agarose Kit
- Antibody Type
- Recombinant Antibody
- Camelid (Camelidae)
- Magnetic Agarose Beads
- Affinity Measurement (AM), Chromatin Immunoprecipitation (ChIP), Enzyme Activity Assay (EAA), Mass Spectrometry (MS), Immunoprecipitation (IP), Protein Complex Immunoprecipitation (Co-IP), Pull-Down Assay (Pull-Down), Purification (Purif)
- RFP-Trap® is a high quality RFP-binding protein coupled to a monovalent matrix (magnetic agarose beads) for biochemical analysis of RFP fusion proteins and their interacting partners.
- Sample Type
- Cell Extracts
- Binding capacity: 10 μL RFP-Trap®_MA slurry binds 3 - 4 μg of RFP
- Cross-Reactivity (Details)
- tested on RFP, mCherry, mOrange, mPlum, tagRFP
- 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.
- RFP-Trap® coupled to magnetic agarose beads
- Material not included
- Lysis buffer (CoIP), 10x RIPA buffer, Dilution buffer, Wash buffer, Elution buffer
- Discover our best selling RFP Primary Antibody
- Application Notes
- Optimal working dilution should be determined by the investigator.
Particle size ~ 40 μm
- Assay Time
- 1.5 h
- - 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 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 2.6
- Sample Collection
- Harvest cells:
For one immunoprecipitation reaction the use of 10^6 - 10^7 mammalian cells (approx. one 10 cm dish) expressing a RFP-tagged protein of interest is recommended. To harvest adherent cells, aspirate growth medium, add 1 mLice-cold PBS to cells and scrape cells from dish. Transfer cells to a pre-cooled tube, spin at 500 g for 3 min at +4 °C and discard supernatant. Wash cell pellet twice with ice-cold PBS, gently resuspending the cells.
1. Resuspend cell pellet in 200 μL ice-cold lysis buffer by pipetting or using a syringe.
note: Supplement lysis buffer with protease inhibitors and 1 mM PMSF (not included). optional for nuclear/chromatin proteins: Use RIPA buffer supplemented with 1 mg/mL DNase, 2.5 mM MgCl2, protease inhibitors and 1 mM PMSF (not included).
2. Place the tube on ice for 30 min with extensively pipetting every 10 min.
3. Centrifuge cell lysate at 20.000x g for 10 min at +4 °C. Transfer lysate to a pre-cooled tube. Add 300 μL dilution buffer to lysate. Discard pellet.
note: At this point cell lysate may be put at -80 °C for long-term storage. optional: Add 1 mM PMSF and protease inhibitors (not included) to dilution buffer
We recommend that during incubation with the beads the final concentration of detergents does not exceed 0.2 % to avoid unspecific binding to the matrix. If required, use more dilution buffer to dilute the supernatant accordingly.
- Assay Procedure
4. Vortex RFP-Trap®_MA beads and pipette 25 μL bead slurry into 500 μL ice-cold dilution buffer. Magnetically separate beads until supernatant is clear. Discard supernatant and repeat wash twice.
5. Add diluted lysate (step 3) to equilibrated RFP-Trap®_MA beads (step 4). If required, save 50 μL of diluted lysate for immunoblot analysis. Tumble end-over-end for 1 hour at 4 °C.
6. Magnetically separate beads until supernatant is clear. If required, save 50 μL supernatant for immunoblot analysis. Discard remaining supernatant.
7. Resuspend RFP-Trap®_MA beads in 500 μL dilution buffer. Magnetically separate beads until supernatant is clear. Discard supernatant and repeat wash twice.
optional: Increase salt concentration in the second washing step up to 500 mM.
8. Resuspend RFP-Trap®_MA beads in 100 μL 2x SDS-sample buffer.
9. Boil resuspended RFP-Trap®_MA beads for 10 min at 95 °C to dissociate immunocomplexes from RFP-Trap®_MA beads. The beads can be magnetically separated and SDS-PAGE is performed with the supernatant.
10. optional instead of steps 8 and 9: elute bound proteins by adding 50 μL 0.2 M glycine pH 2.5 (incubation time: 30 sec under constant mixing) followed by magnetic separation. Transfer the supernatant to a new tube and add 5 μL 1M Tris base pH 10.4 for neutralization. To increase elution efficiency this step can be repeated.
- For Research Use only
- Storage buffer: 20 % EtOH
- Handling Advice
- Do not freeze.
- 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).
- 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).
- Target Name (Antigen)
- Alternative Name
- RFP fluorescent proteins (RFPs) and variants thereof are widely used to study proteinlocalization and dynamics. For biochemical analysis including mass spectrometry and enzyme activity measurements these RFP-fusion proteins and their interacting factors can be isolated fast and efficiently by immunoprecipitation using the RFP-Trap®. RFP-Trap® utilizes small recombinant alpaca antibody fragments covalently coupled to the surface of agarose beads.