1-Step Heavy Protein In Vitro Protein Expression Kits
Express full-length heavy proteins for mass spectrometry proteomics analysis.
Thermo Scientific 1-Step Heavy Protein In Vitro Protein Expression Kits enable rapid cell-free expression of recombinant proteins containing stable isotope-labeled (i.e., heavy) amino acids.
The 1-Step Heavy Protein IVT Kit uses a unique HeLa cell lysate supplemented with heavy amino acids for in vitro translation (IVT) of proteins with 90 to 95% isotope incorporation efficiency in less than 8 hours. Heavy proteins expressed using this system can be used as mass spectrometry controls for sample prep loss, digestion efficiency determination or as quantification standards.
Efficient – express heavy proteins with 90 to 95% stable isotope incorporation
Functional – uses the human translational machinery to express more biologically active proteins than other IVT systems
Flexible – express light proteins or heavy proteins containing 13C615N2 L-lysine and/or 13C615N4 L-arginine
Convenient – perform transcription and translation in a single step
High performance – greater yields compared to rabbit reticulocyte in vitro translation
Identify ideal peptides for targeted quantitation
Verify protein digestion efficiency
Control for protein sample prep variability and affinity enrichment loss
Thermo Scientific 1-Step Heavy Protein IVT Kit contains all of the necessary components to express a heavy protein including HeLa cell lysate, proprietary accessory proteins, reaction mix, heavy amino acids, positive-control GFP DNA and the pT7CFE1-CGST-HA-His cloning vector. The benefits of in vitro expression of heavy proteins over traditional in vivo systems include expression of toxic or insoluble proteins, a more rapid protein synthesis and a more economical use of heavy amino acids compared to stable-isotope labeled cell lines. This small-scale expression method makes it easy to express numerous heavy proteins simultaneously or to express large quantities of a single heavy protein up to 100µg/mL. Tandem affinity purification of heavy proteins is aided using an expression vector containing multiple affinity tags including GST, HA and 6xHis.
Expression and analysis of stable-isotope labeled BAD protein with the Thermo Scientific 1-Step Heavy Protein IVT Kit.A. HA-tagged BAD was expressed using the 1-Step Heavy Protein IVT Kit and affinity purified using the Thermo Scientific Pierce HA Tag IP/Co-IP Kit and analyzed by SDS-PAGE. B. The protein bands were excised, digested and analyzed using a Thermo Scientific LTQ Orbitrap XL mass spectrometer and identified as heavy BAD and light 14-3-3σ which co-purified during immuoprecipitation. MS spectrum of a light and heavy BAD peptide showing >95% heavy isotope incorporation. MS/MS spectrum used for peptide identification is shown in the figure insert.
Heavy Amino Acid Incorporation
Table showing average total average heavy amino acid incorporation into recombinant proteins expressed using the 1-Step Heavy Protein IVT Kit.
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