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Gel electrophoresis
Equipment and consumables for analytical 1D- and 2D-PAGE are available. For 2D separations, the initial step involves isoelectric focusing (IEF) followed by SDS-PAGE or native protein electrophoresis. An integrated package (BIO-RAD PROTEAN IEF cell and vertical slab electrophoresis cells) using pre-cast gels allows simultaneous development of up to twelve 11 cm2 or 17 cm2 gels. Immobilized pH gradient (IPG) strips are available in a variety of pH ranges (from 3-10).
Gel imaging/spot-cutting
A ProteomeWorks spot cutter system with its associated software is used for automated location, excision and transfer of spots into 96-well plates prior to in-gel digestion. Over a dozen different gels can be compared to a master gel(s), and spots chosen for excision, digestion and identification based either on qualitative or quantitative criteria. Both visible and UV imagers are available, allowing use of a variety of staining procedures.
Proteolytic digestion
After spot excision, the 96-well plates to which they are transferred are moved to a Micromass MassPREP robot for automated reduction, alkylation, proteolytic digestion and peptide extraction, followed by spotting on a MALDI target. Approximately 10% of a given digest is used for MALDI analysis, with sufficient sample left for subsequent LC-MS analysis (if necessary) as described below. The robot is easily programmed to accommodate additional steps, such as ZipTip purification, if necessary.
Mass spectrometric analysis, data base searching and protein identification
Digested samples spotted by the MassPREP robot are analyzed using a Micromass M@LDI HT (MALDI-TOF reflectron instrument), and automated data base searches are performed using either ProteinLynx software provided by Micromass, MASCOT (Matrix Science), Protein Prospector (University of California, San Francisco), or PIUMS (Protein identification Using Mass Spectrometry) a new search engine developed by BioBridge Computing (Lund, Sweden).
Proteins not identified by MALDI screening are analyzed using nanospray LC-MS on an Agilent LC/MSD ion trap using capillary LC, followed by acquiring and processing LC-MS data using methods modified by the UCLA facility in conjunction with Agilent personnel. Currently the detection limits are ca.10-100 femtomoles for generating MS/MS data sufficient for unambiguous protein identification.
Two Q-TOF instruments instruments with LC interfaces, one of which also has a MALDI source, are available for analysis of samples requiring higher resolution (ca. 10,000), sensitivity (10 femtomoles/uL) and/or mass range. LC analyses using either the ion trap or the Q-TOF instruments use standard C-18 columns and mildly acidic H2O/acetonitrile gradients for peptide separations., and can easily be adapted to 2-D LC if necessary for more complex mixtures.
The FTICR instrument has both ESI and MALDI interfaces, and is used when extraordinarily high mass accuracy (+/- 2 millimass units) and resolution (50,000-70,000) are required.
Recharge
All services are provided at a flat rate of $87/hour (one hour minimum). There is no charge for data processing if it is performed by the user. All reservations for instrument time must be made using the links provided below under Forms and Reservations.