RFP-Trap Affinity Reagents

Novel Antibody Fragment that binds RFP Fusion Proteins

Analyze RFP fusion proteins – in a tube

Chimeric proteins containing naturally-evolved fluorescent proteins are popular tools to study protein localization and dynamics using microscopy. These constructs usually fuse the entire, or at least a functional domain, of a target protein to one of the multitude of fluorescent proteins. Those originally derived from the mushroom coral Discosoma sp. are commly referred to as red fluorescent protein or RFP. Cellular analysis of the RFP-fusion protein construct is easily accomplished with a host of available live cell imaging solutions. However - to get the full picture - such data have to be combined with additional biochemical information for the respective target proteins, typically fused to a different “tag” domain that allows for purification. These additional in vitro analyses can be used to confirm functionality of the “tagged” fusion construct, as well as to pull out multi-protein complexes that may form in the cellular milieu. So far, the lack of specific, reliable, and efficient reagents has limited the possibility to combine the cell biology results with direct biochemical analysis. Until now, that is.

 



 

 

 

LITERATURE
Protocols
White Paper
FAQs
RFP-Trap References
 
RELATED PRODUCTS
Other Nano-Traps
Nano-Booster Fluorescence  Enhancers
GFP & RFP Antibodies
Live Cell Imaging  Microscopes

 

RFP-Traps utilize super-high affinity Camelidae antibody fragments coupled to agarose beads, magnetic agarose beads, or magnetic particles. These “Nano-Traps” are perfect for immuno-precipitation, immuno-purification and immuno-pull down experiments with up to 10-fold better purity and yield than that of classic mouse monoclonal antibodies. Compatible with a variety of source materials, Nano-Traps may be used with mammalian cells, tissues & organs, bacteria, yeast and even plants. These reagents allow your RFP-fusions to be perfect candidates for immunoprecipitations, Co-IP, mass spectroscopy, enzyme activity measurements, and ChIP analysis.

 

Nano-Traps

 

RFP-Trap Affinity Reagents are perfect for fast, clean and efficient one-step isolation of these fluorescent fusion proteins and their interacting factors. With eight years of production experience and over 100 publications attesting to their broad use and effectiveness, we invite you to try this unique reagent for free. They've become a staple of cell biology research from Europe to North America. The RFP-Traps are offered bound to beads or particles, as well as a free complex, providing you the ability to maximize its use.

 

RFP-Trap Comparison

Nano-Traps allow GFP and RFP to be used as affinity tags

Immunoprecipitations of RFP from human cells expressing RFP. Input (I), non-bound (FT) and bound (B) fractions were separated by SDS-PAGE followed by Coomassie staining and Western Blotting. The bound fraction of the conventional RFP antibodies shows in addition to RFP contaminating heavy chain and light chain. The bound fraction of the RFP-Trap does not contain these contaminating polypeptide chains.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

GFP-Trap Comparison

Immunoprecipitates a wide variety of red fluorescent proteins with RFP-Trap

Pull down of different monomeric red fluorescent protein variants (mRFP, mCherry and mOrange) from human cell extracts. Input (I), non-bound (FT) and bound (B) fractions separated by SDS-PAGE followed by Coomassie staining and Western Blotting.

 

 

 

 

 

 

 

 

 

 

 

GFP-Trap Comparison 2

Protein complexes canít hide from Nano-Traps

To the right are results from using GFP-Trap bound to magnetic particles (GFP-Trap M) and agarose beads (GFP-Trap A). Comparison of GFP-Trap A and GFP-Trap M was first performed with a simple, 1-step, pull-down of native GFP with GFP-Trap A and GFP-Trap M from 293T cell extracts. Input (I) and bound (B) fractions were separated by SDS-PAGE followed by Coomassie staining. As expected, the bound fractions containing GFP are extraordinaly pure. The same 1-step purification procedure was now applied to GFP fused to the protein PCNA in a co-immunoprecipitation type experiment. This time, the chimera and any associated proteins were very efficiently purified with both GFP-Trap A and GFP-Trap M from 293T cell extracts. As can be seen for the gel, the additional bands represent potential binding partners for the GFP-PCNA.

 

 

 

 

What’s your lab’s favorite flavor?

Nano-Traps are high quality binding proteins coupled to a monovalent matrix (agarose beads, magnetic particles or in 96-well plates) for biochemical analysis of many fusion proteins and their interacting partners. The different formats allow you to perform a multitude of experiments. The most popular forms are when the Nano-Traps are linked to agarose beads. Agarose beads provide the largest binding capacity and lowest background, while still being easy to work with. If automation is important to your lab, then magnetic particles and magnetic agarose particles are a great choice. The magnetic particles are smaller and easier to keep in suspension, while the magnetic agarose particles offer the superior binding kinetics. For high throughput, nothing beats the 96-well plate in which the Nanobodies are bound to the side of the wells. These resins can be used interchangeably for pull-down type experiments such as immunoprecipitations, co-IPs and even ChIP assays. The purified protein complexes are compatible with down-stream analyses such as mass spectroscopy.

 

Camelidae Antibody

Camelidae single-domain antibodies are like IgGs on steroids

The family of animals known as Camelidae (camels, dromedaries, llamas and alpacas) produce functional antibodies devoid of light chains, so called "heavy chain" antibodies. These heavy chain antibodies recognize and bind their antigens via a single variable domain. When cleaved from their carboxy tail, these barrel-shaped structures (2x3 nm) are extraordinarily small, naturally-occurring, and intact antigen binding fragments (MW  of 13 kDa). These fragments, called Nanobodies, are characterized by high specificity and affinities in the low nanomolar range, and dissociation constants in the sub-nanomolar range (typically 10- to 100-fold better than mouse IgGs). The compact size of Nanobodies makes them extremely stable at temperatures up to 70°C, and functional even in 2M NaCl or 0.5% SDS. These small and powerful antibody fragments can be used in a variety of unique applications. They will open up your research possibilities.

 

 

 

SPECIFICATIONS

Configuration:

 

RFP-Trap A

Specific Camelidae antibody linked to agarose bead

Part Numbers
RTA010, RTA020, RTA100, RTA200, RTA400, RTAK020

RFP-Trap M

Specific Camelidae antibody linked to magnetic particle (particles from ChemiCell)

Part Numbers
RTM010, RTM020, RTM100, RTM200, RTM400, RTMK020

RFP-Trap MA

Specific Camelidae antibody linked to magnetic agarose particle

Part Numbers
RTMA010, RTMA020, RTMA100, RTMA200, RTMA400, RTMAK020

RFP-Trap

Uncoupled and purified Camelidae antibody

Part Numbers
RT250

Specificity:

 

RFP-Traps

mRFP1, mCherry, mPlum, mOrange, mRFPruby, tdTomato, Stawberry, mKate2

(does not recognize DsRed, TagRFP or all GFPs)

Binding Capacity:

 

RFP-Trap A

10µl binds 3-4µg of recombinant GFP/RFP

RFP-Trap M

10µl binds 0.25-0.5µg of recombinant GFP/RFP

RFP-Trap MA

10µl binds 3-4µg of recombinant GFP/RFP

Particle Size:

 

RFP-Trap A

~90 µm  

RFP-Trap M

0.5 - 1 µm

RFP-Trap MA

~40 µm

RFP-Trap

No particle coupled

Storage Buffer:  

RFP-Trap A

20% EtOH

RFP-Trap M

1x PBS; Preservative: 0.01% Sodium Azide

RFP-Trap MA

20% EtOH

RFP-Trap

1x PBS; Preservative: 0.01% Sodium Azide

Storage and Stability:

 

RFP-Trap A

store at 4°C; stable for one year. Do not freeze.

RFP-Trap M

store at 4°C; stable for one year. Do not freeze.

RFP-Trap MA

store at 4°C; stable for one year. Do not freeze.

RFP-Trap

store at 4°C; stable for one year. Do not freeze.



RFP Traps Coupled to Agarose Beads

ORDERING - RFP-Traps Coupled to Agarose

 

 

RFP Traps Coupled to Magnetic Beads

ORDERING - RFP-Traps Coupled to Magnetic Beads

 

 

RFP Traps Coupled to Magnetic Agarose Beads

ORDERING - RFP-Traps Coupled to Magnetic Agarose Beads

 

 

 

RFP Traps Non-Coupled

ORDERING - RFP-Traps Non-Coupled

 

Binding Controls

 

 

Spin Columns