RFP-Trap Agarose
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I: Input, FT: Flow-through, B: Bound
Description
RFP-Trap® Agarose is an affinity resin for immunoprecipitation of RFP-fusion proteins.
It consists of an RFP Nanobody/ VHH coupled to agarose beads.
Specificity
mCherry, mRFP, mPlum, tagRFP, mKate2 and more RFP derivatives; see:
Fluorescent protein specificity table
Applications
Immunoprecipitation/ Co-IP
Mass spectrometry
On-bead enzyme assays
ChIP/ RIP analysis
| Product | Size | Code | Price | Buy |
|---|---|---|---|---|
| Product RFP-Trap Agarose | Size 250 µL (10 reactions) | Code rta-10 | Price $ 275 |
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| Product RFP-Trap Agarose | Size 500 µL (20 reactions) | Code rta-20 | Price $ 475 |
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| Product RFP-Trap Agarose | Size 2.5 mL (100 reactions) | Code rta-100 | Price $ 2050 |
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| Product RFP-Trap Agarose | Size 5 mL (200 reactions) | Code rta-200 | Price Please inquire | |
| Product RFP-Trap Agarose | Size 10 mL (400 reactions) | Code rta-400 | Price Please inquire | |
| Product RFP-Trap Agarose Kit | Size 500 µL (20 reactions) | Code rtak-20 | Price $ 595 |
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| Product Binding Control Agarose Beads | Size 500 µL (20 reactions) | Code bab-20 | Price $ 75 |
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| Product Spin Columns | Size 10 units | Code sct-10 | Price $ 35 |
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| Product Spin Columns | Size 20 units | Code sct-20 | Price $ 60 |
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| Product Spin Columns | Size 50 units | Code sct-50 | Price $ 130 |
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| Product RFP VHH, recombinant binding protein | Size 250 µL | Code rt-250 | Price $ 275 |
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Affinity
Dissociation constant KD of 5 nM
Wash buffer compatibility
4 M urea, 2 M NaCl, 10 mM DTT, 2 % Nonidet P40 Substitute, 1 % Triton X-100
Specificity (selection)
mRFP, mCherry, mRFPruby, mPlum, tagRFP, mKate2, mOrange, PA-mCherry, mScarlet and more.
For complete list, please click here: Fluorescent protein specificity table
Binding capacity
10 µL slurry bind ~ 9 µg of recombinant mCherry
Coupled Nanobody/ VHH
Recombinant, monoclonal anti-Red Fluorescent Protein (RFP) single domain antibody (sdAb) fragment
Bead properties
Bead size: ~ 90 µm (cross-linked 4% agarose beads)
Storage buffer: 20 % EtOH
Elution
- SDS sample buffer
- 0.2 M glycine pH 2.5
Instead of elution, we recommend on-bead assays like on-bead digestion for MS analysis.
Compatibility with mass spectrometry
The RFP-Trap is optimized for on-bead digestion. Complete tryptic digest results in 6-7 peptides.
How to cite this product
RRID: AB_2631362
Storage instructions
Shipped at ambient temperature. Upon receipt store at 4°C. Stable for one year.
Protocol
Brochure
Trouble shooting
SDS
RFP-Trap Specificity
Application notes
Rose Willett,Jose A Martina,James P Zewe,Rachel Wills,Gerald R V Hammond,Rosa Puertollano (2017), TFEB regulates lysosomal positioning by modulating TMEM55B expression and JIP4 recruitment to lysosomes., Nat Commun, link
Anne Ramat,Matthew Hannaford,Jens Januschke (2017), Maintenance of Miranda Localization in Drosophila Neuroblasts Involves Interaction with the Cognate mRNA., Curr Biol, link
Marie Maclennan,Marta Garcia-Cañadas,Judith Reichmann,Elena Khazina,Gabriele Wagner,Christopher J Playfoot,Carmen Salvador-Palomeque,Abigail R Mann,Paula Peressini,Laura Sanchez,Karen Dobie,David Read,Chao-Chun Hung,Ragnhild Eskeland,Richard R Meehan,Ol. (2017), Mobilization of LINE-1 retrotransposons is restricted by Tex19.1 in mouse embryonic stem cells., Elife, link
Nagendra Babu Thillaiappan,Alap P Chavda,Stephen C Tovey,David L Prole,Colin W Taylor (2017), Ca2+signals initiate at immobile IP3receptors adjacent to ER-plasma membrane junctions., Nat Commun, link
Wei Jiang,Yuehua Wei,Yong Long,Arthur Owen,Bingying Wang,Xuebing Wu,Shuo Luo,Yongjun Dang,Dengke K Ma (2018), A genetic program mediates cold-warming response and promotes stress-induced phenoptosis in C. elegans., Elife, link
Hui Li,Nan Luo,Weidong Wang,Zengyu Liu,Jisheng Chen,Liangtao Zhao,Li Tan,Chunyan Wang,Yuan Qin,Chao Li,Tongda Xu,Zhenbiao Yang (2018), The REN4 rheostat dynamically coordinates the apical and lateral domains of Arabidopsis pollen tubes., Nat Commun, link
Sheng Sun,Vikas Yadav,R Blake Billmyre,Christina A Cuomo,Minou Nowrousian,Liuyang Wang,Jean-Luc Souciet,Teun Boekhout,Betina Porcel,Patrick Wincker,Joshua A Granek,Kaustuv Sanyal,Joseph Heitman (2017), Fungal genome and mating system transitions facilitated by chromosomal translocations involving intercentromeric recombination., PLoS Biol, link
What are the biophysical parameters of the RFP-Trap®?
Molecular weight: 14,9 kDa; Extinction coefficient: 30035 M-1 cm-1
Should I use an N-terminal or C-terminal fusion tag?
Both the N-terminal or C-terminal fusion tag work well with the traps.
How can I avoid unspecific protein interactions binding to the trap?
For preclearing of your sample we recommend to use our binding controls (bab-20 or bmab-20).
Please find more information in our Troubleshooting guide
How can I elute bound proteins from a trap in their native state?
You can elute your fusion protein of interest with 0.2 M glycine pH 2.5 at room temperature. Pipette the beads up and down for 60-120 seconds and repeat this step. Ensure to neutralize your supernatant immediately afterwards by adding 1 M Tris base pH 10.4.
How many mammalian cells are required for an immunoprecipitation reaction?
For one immunoprecipitation reaction, we recommend using ~10^6 - 10^7 mammalian cells. The yield is also dependent on the expression level of your protein of interest and the interaction partners.
How much cell extract should I use for an immunoprecipitation reaction?
For other type of cells than mammalian cells, we recommend using 0.5 - 1.0 mg of cell extract.
Do I need to elute bound proteins from the beads for mass spectrometry analysis?
No, you can directly conduct an on-bead digestion after immunoprecipitation. This procedure allows faster and more efficient sample preparation and a potential higher yield.
Please find more information here:
Do I need to elute my protein of interest from the beads for enzymatic assays?
No, you can directly perform your enzymatic assay on the beads if the active center is not blocked.
Please find more information in our application note "enzymatic activity assay"
What are the dissociation constants of the Nano-Traps?
Generally heavy chain antibodies do have high affinities to their antigens with dissociation constants in the low nanomolar down to the picomolar range. ChromoTek has determined the following KD values:
GFP-Trap: 1 pM, picomolar (10-12 molar)*
RFP-Trap: 5 nM, nanomolar (10-9 molar)
MBP-Trap: 4 nM, nanomolar (10-9 molar)
GST-Trap: 1 nM, nanomolar (10-9 molar)*
Myc-Trap (with 2x Myc peptide): 0.5 nM, nanomolar (10-9)
Spot-Trap: 6 nM, nanomolar (10-9 molar)
*Kinetic parameter has been measured using the switchSENSE technology using electro-switchable nanolevers to analyze molecular interactions. switchSENSE is a proprietary technology from Dynamic Biosensors (www.dynamic-biosensors.com).
What is the amount of trap slurry I need for one immunoprecipitation reaction?
25 µL slurry are sufficient for one pull-down reaction as the affinity of the traps is very high.
Specifications of RFP-Trap Agarose, Magnetic Agarose, and Dynabeads
| RFP-Trap | ||
| Agarose | Magnetic Agarose | Dynabeads |
Matrix | Agarose (4% cross-linked) | Magnetic agarose (6% cross linked) | Dynabeads M-270 |
Bead form | Porous | Porous; sold iron core | Solid |
Ligand | RFP VHH | RFP VHH | RFP VHH |
RFP-tagged protein size* | Small to large size | Small to large size | Small to very large size; no size limitation |
Color | White | Black | Brown |
Medium particle size | 90 µm | 40 µm | 2.8 µm |
Binding capacity | 9 µg/ 10 µL | 8 µg/ 10 µL | 0.5 μg/ 10 μL |
Background | Very low | Low | Low |
Magnetic separation & automation | No | Yes | Yes |
May be centrifuged up to | 2,500 x g | 800 x g | 8,000 x g |
* Does depend on protein size and shape, protein multimers, complexes and interaction partners
Benefits
- Robust performance with clean results
- Short incubation time (30 min)
- No heavy & light chains in your downstream application
- Strong binding, also under harsh washing conditions
RFP-Trap Agarose Kit
The RFP-Trap Agarose is also available in a kit, including:
- RFP-Trap Agarose
- Lysis*, wash and elution buffers
*The lysis buffer provided is suitable for mammalian cells. For other cell types use another suitable lysis buffer.
Spin Columns
- Easy and convenient handling
- Rapid washing and clean elution of bound proteins
- Simplify the pulldown of your protein
- Spin Columns for RFP-Trap Agarose
Binding Control for Agarose Beads
- Control for unspecific binding of proteins, DNA, etc. to beads
- Pre-clearing of cell lysate
- Binding Control for RFP-Trap Agarose
Related products
Testimonials
"...the beads were glowing purple so we knew there was a ton of mCherry on them! I think the RFP-Trap is a really nice reagent..."
Prof. Dr. Laura Trinkle-Mulcahy, University of Ottawa
"We have found your trap tools extremely useful, the purification effect is way superior to most of our antibodies. This tool proved very helpful in our research and we are practically addicted on it."
Dr. Ondrej Plihal, Institute of Microbiology, Department of Ecology
"I am a big fan of all GFP/RFP-Trap for Co-IP experiments and we use it regularly. Same thing for the labeled antigen specific nano-bodies."
Dr. Harald Wodrich, University Bordeaux
Only for research applications, not for diagnostic or therapeutic use!