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Plant Molecular Biology
In vitro Director System
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| In vitro Director provides a unique ligation mediated PCR method to generate DNA templates for coupled in vitro expression systems such as wheat germ and rabbit reticulocyte. The system utilizes a proprietary blend of ExoClone™ dNTPs that result in controlled directional ligation of Anchor™ sequences to any gene. Anchors provide necessary sequences such as the T7 promoter, Kozak ribosomal binding site, and affinity tags. The combination of In vitro Director with a coupled in vitro expression system and downstream 96-well affinity purification products creates a powerful system for high throughput protein expression and characterization that is unmatched in speed and reliability. |
Applications:
- Protein-protein interaction
- Protein-DNA interaction
- Mutation Analysis
- Protein characterization
- Labeling
- Alternative for difficult in vivo expression
– Eukaryotic genes
– Insoluble protein
– Protein is toxic to host cell
– Rapid proteolytic degradation
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Available In vitro Director System products
Target Gene to Protein in Less than a Day
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First round PCR to amplify target gene from cDNA or plasmid DNA in the presence of dNTPαS (2-3 hours) |
Controlled exonuclease III treatment for generation of cohesive ends (10 minutes) |
Directional ligation of 1st round PCR product to Anchors (30 minutes) |
Second round PCR to amplify expression ready DNA templates using universal Anchor Primers (2-3 hours) |
In vitro protein synthesis (1-2 hours) |
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Rapid Assembly of Expression Ready DNA Templates
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In vitro Director Method
- Robust, high throughput assembly
- Validated for >100 genes ranging in size from 239 to 2207 bp
- No concern of internal cutting sites or rearrangements
- No cloning, no transformation, no cell culture
- Excess first Round PCR product can be rapidly cloned using Director-Ready™ Vectors
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Figure 1. Strategy for IκB-α binding domain mapping experiment. Gene-specific primers containing recommended 5 base overhangs were designed to produce full length IκB-α and 5 truncated versions (serial deletions). |
Figure 2. Rapid assembly of IκB-α expression ready templates. Lanes 2-7 are the 630 bp-954 bp first round PCR products amplified from an IκB-α-CMV-2 vector using gene-specific primers (see figure 1) and ExoClone™ dNTP mix. After rapid digestion with exonuclease III to produce cohesive ends, purified products were ligated to the 5' c-Myc Anchor™ set. Assembled templates were amplified using universal Anchor Primers to produce expression ready templates (lanes 10-15) containing the T7 promoter, Kozak sequence, c-Myc tag, etc. |
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One-Step Coupled In vitro Protein Synthesis
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| Figure 3. In vitro expression of IκB-α serial deleted fragments. Expression ready templates produced during second round PCR in Figure 2 were used directly in a coupled rabbit reticulocyte expression system. The synthesized c-Myc-IκB-α fragments were purified from the rabbit reticulocyte lysate through a short incubation on Anti-c-Myc 96-well affinity capture plates, eluted and detected on a western blot using monoclonal Anti-c-Myc and a rabbit Anti-mouse-HRP secondary antibody (chemiluminescent detection). The 210-318 amino acid fragments are shown in lanes 1-6 above. |
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Transcription/Translation of Expression Ready Templates
- Contain T7 promoters, Kozak consensus ribosomal binding sites, and optional affinity tags through ligation with Anchors
- 2nd PCR provides enough template for up to 10 transcription/translation reactions
- Compatible with coupled, uncoupled or linked transcription/translation systems
- Expression validated in wheat germ and rabbit reticulocyte systems
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High Throughput Protein-Protein Interaction Studies
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Analysis of In Vitro Synthesized Protein
- Bait/prey proteins for protein-protein interaction studies can be in vitro synthesized or endogenous protein
- ANTI-FLAG® and Anti-c-Myc 96-well affinity capture plates provide a further streamlined high throughput platform for quantitative analysis
- MALDI, ELISA or Western blotting may be used for analysis
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| Figure 4. Binding Domain Mapping of IκB-α. FLAG-p65, expressed in Cos-7 cells, was captured on an ANTI-FLAG 96-well plate. The anchored FLAG-p65 was used to capture interacting proteins from the transcription/translation reactions in Figure 3. Anti-c-Myc-AP/pNPP was used to detect any interacting c-Myc-IκB-α fragments. According to the ELISA results, a necessary domain for interaction with p65 maps to the region between 252 aa and 277 aa of IκB-α, narrowing the area for future studies to a 25 amino acid region. The entire experiment (target DNA amplification to ELISA results) was completed in only 1.5 days. |
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Universal for Plant and Other Eukaryotic Genes
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Figure 5. Protein-Protein Interaction of Arabidopsis Proteins. Expression Ready templates for Arabidopsis histone-3 (H3) and histone-4 (H4) proteins were prepared using the In vitro Director 5'-c-Myc and 5'-FLAG Anchor Kits. In vitro expression was performed using the Wheat Germ TNT® System. In vitro synthesized c-Myc-H3 and c-Myc H4 (bait proteins) were captured on Anti-c-Myc 96-well plates and incubated with corresponding FLAG-tagged prey proteins (H3 or H4). Protein-protein interaction was detected using ANTI-FLAG-M2-AP conjugate/pNPP. FLAGBAP was used as a negative control. The left panel shows specific interaction of FLAG-H4 with c-Myc-H3 while the right panel shows specific interaction of FLAG-H3 with c-Myc-H4. |
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TNT® T7 Coupled Wheat Germ Extract System TN0100
A Proven System for Director™ Generated Expression Ready Templates
- Easy to use – One-tube coupled transcription/translation
- Robust – Produces 2-6 fold more protein than standard in vitro translation reactions
- Fast – Reaction is completed in only 1.5 hours
- Diverse – Translated protein may be used in a wide variety of applications
- Convenient – Utilizes widely used T7 promoter
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