based on a single domain antibody derived from Lama alpaca.

Chromotek-GFP-Trap, a GFP-binding protein

A genius for quantitative immunoprecipitation of GFP fusion proteins.
Box 1 | Tags for Immunoprecipitation

reen uorescent proteins (GFP) and variants thereof are widely used to study protein localization and dynamics. However, among the most commonly used tags for immunoprecipitation (a brief review in Box 1), the use of GFP is limited due to previously available anti-GFP antibodies, either polyclonal or monoclonal, not being comparable to those against other tags. GFP-Trap is a high quality GFPbinding protein based on a single domain antibody derived form Lama Alpaca. It is characterized by a small barrel shaped structure (13 KDa, 2.5 nm X 4.5 nm) and a very high stability (stable up to 70C, functional within 2 M NaCl or 0.5% SDS). From detailed in vitro binding analysis, we determined that one molecule GFP-Trap binds one molecule GFP in a stable stoichiometric complex with a dissociation constant (Kd) of 0.59 nM. After coupling to monovalent matrices (e.g. agarose beads or magnetic particles), GFP-Trap can be a robust

To achieve effective immunoprecipitation, a researcher must rst overcome the difculty of nding usable antibodies against a target of interest. Using tags that are fused to the C- or N-terminus of the target protein is common practice. In general, while being mindful of the unique nuances with each biological system, choosing tags that have been tested in many situations and been proven to be non-interfering is ideal. The most commonly used tags are as follows: FLAG, Myc, HA, V5, T7, and His; these are quite small in size and in theory less likely to interfere. GST and GFP are well documented to form self-contained and stable structures independent of their fusion partners and proven to not interfere in many cases despite their larger size (in between 20-30kD). A top choice for pulldown experiments, GST can bind to glutathione beads directly. GFP/RFP and their variants are excellent tags, having the advantage of being a visualization module to follow the protein both inside cells and during pulldown. With much greater stability, specicity, and afnity than previously available polyclonal or monoclonal anti-GFP/RFP antibodies, GFP-Trap and RFP-Trap, the recent addition to antibodies for immunoprecipitation should make GFP/RFP the most suitable tag for immunoprecipitation assays.

tool for: * Identify interacting proteins * Measure enzyme activity * Determine DNA or RNA binding * Map DNA or RNA binding sites * ChIP-CHIP assays * RIP assays (RNA immunoprecipitation) * CLIP assays (in vivo Cross-Linking and ImmunoPrecipitation) With much greater stability, specicity, and afnity, GFP-Trap, the recent addition to antibodies for immunoprecipitation, should make GFP the most suitable tags for immunoprecipitation assays. Direct comparison of the GFPTrap with conventional antibodies is shown in Box 2. Besides the original avGFP from jelly sh, GFP-Trap can also bind to the GFPS65T and eGFP versions and as well as YFP and eYFP. It recognizes and binds a three dimensional epitope at the beta barrel structure. GFP-Trap does not recognize unfolded or denatured GFP (e.g. on Western blots). DsRed, which is from coral instead of jellysh, is not recognized by GFPTrap. For binding DsRed-derived FPs such as the commonly used mCherry, mOrange, mPlum, mRuby and mRFP, RFP-Trap should be used.

Box 2 | Comparative Immunoprecipitation Assay

1) GFP-Trap allows a very fast (~ 5 30 min) depletion of GFP from tested samples, which cannot be achieved with conventional antibodies even after 12 h of incubation. 2) Only the antigen (GFP) was detectable on a coomassie gel , there is no heavy chain and light chain

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Product List and Content

GFP-Trap Beads and RFP-Trap Beads: A VHH domain binding protein derived from camelid heavy chain-only antibodies is coupled to agarose or magnetic beads for immunoprecipitation. Typically, 1ul resin can effciently bind 1-3 g proteins. Four unit sizes (0.25 ml, 0.5 ml, 2.5 ml and 5 ml) are available. 0.5 ml size is enough for 20 RXNs. GFP-Trap Kits and RFP-Trap Kits: In addition to the VHH domain binding protein derived from camelid heavy chain-only antibodies being coupled to agarose or magnetic beads for immunoprecipitation, precipitation buffers and a monoclonal anti-GFP antibody are provided for user convenience (0.5ml beads + 10ml lysis buffer + 50ml dilution buffer + 20ml wash buffer (NaCl 500mM) + 100g monoclonal anti-GFP antibody), which is enough for 20 RXNs. GFP-Trap Proteins and RFP-Trap Proteins: Uncoupled VHH domain binding protein derived from camelid heavy chain-only antibodies is provided for custom-conjugation or other assays. The concentration is 1 g/l.
GFP-Trap Beads
GFP-Trap Beads (Agarose) GFP-Trap Beads (Agarose) GFP-Trap Beads (Agarose) GFP-Trap Beads (Agarose) GFP-Trap Beads (Magnetic) GFP-Trap Beads (Magnetic) GFP-Trap Beads (Magnetic) GFP-Trap Beads (Magnetic) ACT-CM-GFA0025P ACT-CM-GFA0050 ACT-CM-GFA0250 ACT-CM-GFA0500 ACT-CM-GFM0025P ACT-CM-GFM0250 ACT-CM-GFM0500 ACT-CM-GFM0500 0.25ml 0.5ml 2.5ml 5.0ml 0.25ml 0.5ml 2.5ml 5.0ml

RFP-Trap Beads
RFP-Trap Beads (Agarose) RFP-Trap Beads (Agarose) RFP-Trap Beads (Agarose) RFP-Trap Beads (Agarose) RFP-Trap Beads (Magnetic) RFP-Trap Beads (Magnetic) RFP-Trap Beads (Magnetic) RFP-Trap Beads (Magnetic) ACT-CM-RFA0025P ACT-CM-RFA0050 ACT-CM-RFA0250 ACT-CM-RFA0500 ACT-CM-RFM0025P ACT-CM-RFM0050 ACT-CM-RFM0250 ACT-CM-RFM0500 0.25ml 0.5ml 2.5ml 5.0ml 0.25ml 0.5ml 2.5ml 5.0ml

GFP-Trap Kits
GFP-Trap Kit (Agarose) GFP-Trap Kit (Magnetic) ACT-CM-GFAK020 ACT-CM-GFMK020 ACT-CM-GFPTRAP 20 RXNs 20 RXNs 250l, 1g/l

RFP-Trap Kits
RFP-Trap Co-IP Kit (Agarose) RFP-Trap Co-IP Kit (Magnetic) ACT-CM-RFAK020 ACT-CM-RFMK020 ACT-CM-RFPTRAP 20 RXNs 20 RXNs 250l, 1g/l

GFP-Trap Protein

RFP-Trap Protein

GFP-Trap Booster: A VHH domain binding protein derived from camelid heavy chain-only antibodies is coupled to a strong uorescent dye to both stabilize GFP and enhance its uorescence signals.
GFP-Trap Booster ABP-CM-GBOOSTR 100ug

FP-TrapTM Binding Control Blocked agarose beads or blocked magnetic particles, as binding controls.
FP-TrapTM Binding Control
Blocked agarose beads Blocked magnetic particles ACT-CM-BDCTRLA ACT-CM-BDCTRLM 0.5ml 0.5ml

Conventional Anti-GFP/RFP antibodies: Monoclonal or polyclonal antibodies against GFP/RFP are provided for post-precipitation detection or as controls.
Conventional Anti-GFP Antibodies
Mouse monoclonal antibody to GFP Rabbit polyclonal antibody to GFP Rat monoclonal [3H9] to GFP Rat monoclonal [5F8] to RFP ABP-MAB-GT007 ABP-PAB-PAGFP10 ACT-CM-MRGFP10 ACT-CM-MRRFP10 100ug, IgG1, for WB, ELISA, IP 100ug, IgG, for WB, ELISA, IP 100ug, IgG2a , for WB, ELISA, IP 100ug, IgG2a, for WB, ELISA, IP

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Protocols
Storage: at 2-8C, do not freeze. Cell Lysis 1. For one immunoprecipitation reaction resuspend cell pellet (~10^7 cells) in 200l lysis buffer by pipetting (or using a syringe) 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 4C 4. Transfer supernatant to a pre-cooled tube. Adjust volume with dilution buffer to 500l1000l. Discard pellet The cell lysate can be frozen at this point for long-term storage at minus 80C. For immunoblot analysis dilute 50l cell lysate with 50l 2x SDS-sample buffer. (-> refer as input) Technical Notes: Different buffers can be used to disrupt cells, for exmaple higher salt or DNase I containing buffer to release chromatin proteins; RIPA buffer to release membrane bound proteins ; different concentrations and combinations of zwitterionic detergents to further optimize the sample preparation. Compared to commonly used ionic detergent such as SDS, or non-ionic detergent such as Triton X-100, a less know group called zwitterionic detergents contain both a positive and negative charge in their hydrophilic head group. These compounds are electrically neutral like the nonionic detergents, but can efciently disrupt most protein-protein interactions like the ionic detergents. For co-IP, non-specic protein aggregation is to be disrupted, whereas specic, stronger protein-protein interactions are to be preserved. By using different concentrations and combinations of zwitterionic detergents, or sugar-derived polar head-containing detergents, efcient cell lysis and protein extraction may be achieved for co-IP. However, there is unfortunately no one solution that works well for all cell types, proteins, or coIP requirements. A common buffer as recommended in the GFP-Trap protocol, or an available system like AlleleExtractM should provide you with a good starting point. GFP-Trap Binding 5. Equilibrate GFP-Trap beads in dilution buffer. Resuspend 20 - 30l Beads Slurry in 500l ice cold dilution buffer and spin down at 2700x g for 2 minutes at 4C. Discard supernatant and wash binder two more times with 500l ice cold dilution buffer. 6. Add cell lysate to equilibrated GFP-Trap_A beads 7. Incubate with gentle end-over-end mixing for 10 min 2 h at room temperature or 4C 8. Spin tube at 2000x g for 2 minutes at 4C 9. For western blot analysis dilute 50 l supernatant with 50 l 2x SDS-sample buffer (-> refer as non-bound) 10. Discard remaining supernatant 11. Wash pellet two times with 500 l ice cold wash buffer (Optional: increase salt concentration in the second washing step up to 500 mM) Technical Notes: Binding Capacity: typically 1ul resin can effciently bind 1-3ug proteins. The actual binding amount will depend on different proteins. Elution 12. Resuspend GFP-Trap_A beads in 100 l 2x SDSSample buffer 13. Boil resuspended beads for 10 minutes at 95C to dissociate the immunocomplexes from the beads. The beads can be collected by centrifugation at 2700x g for 2 minutes at 4C and SDS-PAGE is performed with the supernatant. (-> refer as bound) Alternative: elute bound proteins by adding 50 l 0.1 M glycine pH 2.5 (incubation time: 30 sec 2 min) followed by neutralisation with 5 l 1M Tris-base. Technical Notes: To elute native complex, the best way is acidic elution using 0.1 M glycine pH2.5 combined with a very fast addition of 1 M Tris-base for neutralization.

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Suggested Buffers (as tested in our laboratory)

Lysis-buffer (for CoIP): 10 mM Tris/Cl pH7.5 150 mM NaCl 0.5 mM EDTA 0.5% NP40 1 mM PMSF freshly added (optional) 1x mammalian Protease Inhibitor Cocktail (e.g. Serva) freshly added (optional for nuclear proteins / chromatin proteins: DNaseI nal conc. 1 g/l 2.5 mM MgCl2) Dilution-buffer 10 mM Tris/Cl pH7.5 150 mM NaCl 0.5 mM EDTA 1 mM PMSF freshly added (optional) 1x Protease Inhibitor Cocktail (e.g. Serva) freshly added Wash-buffer 10 mM Tris/Cl pH7.5 150 - 500 mM NaCl 0.5 mM EDTA 1 mM PMSF freshly added (optional) 1x Protease Inhibitor Cocktail (e.g. Serva) freshly added RIPA-Buffer (for cell lysis): 10 mM Tris/Cl pH7.5 150 mM NaCl 0.1% SDS 1% TX100 1% Deoxycholate 5 mM EDTA 1 mM PMSF freshly added (optional) 1x Protease Inhibitor Cocktail (e.g. Serva) freshly added

Camelid Antibody
amelidae sp. family animals such as Camel and Llama generate antibodies composed of heavy chains only. The variable domain of camelid antibody heavy chain is the smallest single domain antigen-binding fragment. The ~15kD fragment, term nanobody can recognize specic antigens extremely well and bind very tightly. Combined with its small size and ease for production in E. coli, the single domain Camelid antibody fragment presents unprecedented possibilities in live cell imaging as well as target molecule isolation. Generating camelid antibodies starts with creating a display library of variable domain VHH from immunized Llama, then screening displaying phages or cells to nd clones that express nanobodies against the specic antigen with high afnity and specicity, and cloning the gene encoding the antigen-binding fragment to be expressed in E. coli. Smaller antibody fragments have been tested for therapeutic uses since classical IgG antibodies are too large to penetrate tissues well and are costly to produce. Different combinations of antigen-binding variable regions have been used (e.g., scFv, Fab, diabody) with varying degree of success. By comparison, the N-terminal domain of camelid antibodies, termed VHH domain (nanobody), represents a naturally evolved, fully functional target binding fragment with many advantages-one being that it is only 13-15 kD in size.

The only other known species outside the camelid family that has heavy chain alone antibodies is the cartilaginous nurse shark. Although the arrangement of CDRs is somewhat different in the camel and shark heavy chain variable regions, they share many characteristics, including extremely high stability. They remain functional after 100?C heat and extreme pH treatment. Accumulating reports have demonstrated the therapeutic potentials of camelid antibody-based fragments in treating cancer and neural diseases. They can even be used in shampoo to prevent dandruff. For basic research, these tiny antigen binders can result in quantitative pull down (immunoprecipitation or co-immunoprecipitation, co-IP) with unmatched efciency. Nanobodies can also bind to previously inaccessible protein structure clefts such as enzyme active sites, penetrate in vivo barriers, and provide libraries for binding partner selection. Allele Biotech has been working on display antibody selection since its early days through an NIH grant. We carried out a NIH/NCI contract for scFv yeast display in 2008 in collaboration with our partners. In 2009 Allele Biotech is introducing camelid antibody based products to the US market by working with Chromotek.

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