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.
Highlights:
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
Applications:
Identify ideal peptides for targeted quantitation
Verify protein digestion efficiency
Control for protein sample prep variability and affinity enrichment loss
Product Details:
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.
Expressed Protein
Heavy Amino Acid Incorporation
BAD
92%
CCND1
97%
TP53
91%
RB
96%
GAPDH
94%
GFP
95%
Table showing average total average heavy amino acid incorporation into recombinant proteins expressed using the 1-Step Heavy Protein IVT Kit.
Mikami, S., et al. (2006). An efficient mammalian cell-free translation system supplemented with translation factors. Protein Expr. Purif. 46(2):348-57.
Mikami, S., et al. (2008). A human cell-derived in vitro coupled transcription/translation system optimized for production of recombinant proteins. Protein Expr. Purif. 62(2):190-8.
Hanke S, et al. (2008). Absolute SILAC for accurate quantitation of proteins in complex mixtures down to the attomole level. J. Proteome Res.7(3):1118-30.
Ciccimaro E., et al. (2009). Absolute quantification of phosphorylation on the kinase activation loop of cellular focal adhesion kinase by stable isotope dilution liquid chromatography/mass spectrometry. Anal. Chem. 81(9):3304-13.
Stergachis, A., et al. (2011) Rapid empirical discovery of optimal peptides for targeted proteomics. Nat. Meth. 8(12):1041-1043.
