With the completion of the human genomic sequences, biological researchers are turning to the systemic analysis of biological systems. A complete description of a biological system requires analysis on both the nucleic acid and protein levels. Major efforts of current proteomic analysis technologies are directed toward the rapid and sensitive identification of the proteins that control biological systems. However, rapid and accurate identification and characterization of large numbers of proteins remain significant challenges in proteomics research. One of the most commonly used methods for protein analysis and characterization is 2D gel electrophoresis, followed by in-gel digestion and mass spectrometric analysis of the resulting peptides. The ICAT™, isotope coded affintiy tag, method followed by 2-D HPLC allows quantitative characterization of cysteine containing peptides. Similar methods are also being developed for use on proteins in solution. The mass spectrometric fingerprints are subsequently used in the search of sequence databases for the identification of proteins. Success in the identification may vary with the completeness of the database, the sensitivity of the mass spectrometer, the presence of isobaric masses, post-translational modifications, etc.
Benefits of Pi3™ Peptide Reagents
The Pi3 PhosphoTyrosine-QE Reagent Kit is the first in a family of phosphate specific reagent kits that provides an easy self-contained method, using a novel water-insoluble reagent that forms a covalent bond with phosphotyrosine peptides. The reagent allows for the selective isolation of PhosphoTyrosine peptides from peptide mixtures, or for the collective isolation of pSer, pThr and pTyr, down to the 2 percent occurrence level. These separated phosphate containing peptides subsequently may be regenerated and analyzed by mass spectrometry or liquid chromatography. Use of the mass spectrometric data and the method of digestion, when coupled with the added constraint that the peptides contain PhosphoTyrosine, can lead to improved accuracy in the identification of proteins. Chromatographic analysis of the PhosphoTyrosine peptides can simplify complex separations.
The Pi3 PhosphoTyrosine-QE Reagent is a solid-phase reagent which performs the following chemistry on a phosphotyrosine peptide:
The Pi3 PhosphoTyrosine-QE Reagent is a solid-phase reagent which performs the following chemistry on pSer, pThr and pTyr peptides:
The method employed for the use of the Pi3 PhosphoTyrosine-QE Reagent consists of 5 basic steps using standard laboratory reagents:
1. Capture the phosphotyrosine peptides (or pSer, pThr and pTyr as a group) on the solid-support.Equipment Required
2. Wash the support to remove any non-bound peptides.
3. Release the bound phosphotyrosine peptides.
4. Collect the released peptides and wash the solid-support.
5. Desalt and concentrate the phosphotyrosine peptide solution prior to analysis.
In addition to pipettors, balances, etc., the following equipment is needed for the use with the Pi3 PhosphoTyrosine-QE Reagent Kit:
1. Vortex mixer with micro tube insert:
a. The holes in the insert must be, or must be modified to be ~.375" (10mm) in diameter
b. If the holes in the insert are not through holes, the depth of the holes must be ~1.75" (45mm) deep.
c. The Vortex Genie II, fitted with the 6" platform head and the 60 micro tube insert is recommended.
2. Bench top centrifuge:
a. Capable of 10,000 RPM
b. Capable of holding 1.5-2 ml microcentrifuge tubes
Pi3™ PhosphoTyrosine-QE Reagent Kit Part No. X-7204-4 Price: $200.00
Pi3™ PhosphoTyrosine-QE Reagent Kit Part No. X-7204-8 Price: $350.00
Included are:Online Order Form
1. Four or Eight isolation packs, respectively.
2. One accessory pack
Capacity of each isolation pack is for a digestion of up to 5 nmol of protein.
Each Pi3 PhosphoTyrosine-QE Reagent kit contains material enough for four or eight isolation reactions, respectively.
Last Updated: 01/19/18
ICAT™ is a trademark of the University of Washington.