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A well accepted “Gold Standard” in proteomics, 2-D Electrophoresis proteomics differential display analysis of protein samples followed by protein species isolation and sequencing by mass spectrometry, is offered as a service:
1. Gradient strips with pH ranging from 4 to 7 are loaded via in-gel rehydration.
2. First-dimension isoelectric focusing is performed at 95,000 Volt-hours.
3. Second-dimension separation is done using large-format polyacrylamide slab gels with 8% to 18% thymine for 6 hours at 3 mA per gel and for 14 hours at 15 mA per gel.
4. Gels are stained with SYPRO Ruby fluorescent stain.
5. All gel images are scanned and compared using Z3 Image Analysis software (Compugen, Onatario, Canada).
6. Protein bands of interest are excised and isolated from 2-D gels and are subjected to a 16-hour tryptic digest at 37 degrees Celsius.
7. The resulting peptides are purified, desalted, and concentrated.
8. The samples are analyzed by Electrospray Ionzation- Time-of-Flight Tandem Mass Spectrometry (ESI-QTOF-MS/MS) using a Micromass Quadrupole Time-of-Flight mass spectrometer equipped with a nanospray source, using either flow injection coupled to a Waters CapLC column (Waters Corp., Melford, MA) or manual acquisition using borosilicate capillaries for nanospray acquisition.
9. Data is acquired over the m/z range of 400-1600 to select peptides for tandem mass spectrometry analysis. After peptides are selected, the mass spectrograph is switched to tandem mass spectrometry mode, and data is collected over the m/z range 50-2000 with variable collision energy settings.
10. The data is compared against the OWL database using the MASCOT search engine(http://www.matrixscience.com) with the species limitation and is compared against the SWISS-PROT database using ProteinLynx.
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