RFP-Trap Affinity Reagents

Novel Antibody Fragment that binds RFP Fusion Proteins

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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 entirety–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 commonly 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. –To get the full picture, though, 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 vitroanalyses 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 combination of cell biology results with direct biochemical analysis. Until now, that is.

RFP-Traps utilize super-high affinity Camelidae antibody fragments coupled to agarose beads or magnetic agarose beads. These “Nanobody-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, Nanobody-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

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 throughout Europe and North America.


Nanobody-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 heavy chain and light chain contaminants in addition to RFP. The bound fraction of the RFP-Trap does not contain these contaminating polypeptide chains.


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.

 


Protein complexes can’t hide from Nanobody-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 extraordinarily pure. The same 1-step purification procedure was then 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 from the gel, the additional bands represent potential binding partners for the GFP-PCNA.


What’s your lab’s favorite flavor?

Nanobody-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 Nanobody-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 single-domain antibodies are like IgGs on steroids

The family of animals known as Camelidae (camels, 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, 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.


RFP-Traps Coupled to Agarose

RFP-Traps Coupled to Magnetic Agarose Beads

RFP-Traps Non-Coupled

Binding Controls

Spin Columns

SPECIFICATIONS
Configuration:

RFP-Trap A

Specific Camelidae antibody linked to agarose bead
Part Numbers
RTA010, RTA020, RTA100, RTA200, RTA400, RTAK020

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

mRFP, mCherry, mPlum, mOrange, mRFPruby, mRuby2, mKate2, PA-mCherry, TagRFP

(weak binding to DsRed1 and tdTomato)

Binding Capacity:

RFP-Trap A

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

RFP-Trap MA

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

RFP-Trap A

~90 µm

RFP-Trap MA

~40 µm

RFP-Trap

No particle coupled
Storage Buffer:

RFP-Trap A

20% EtOH

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 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.