The Protein Chemistry Core offers custom peptide synthesis and purification utilizing both natural and non-natural amino acids. Modifications such as biotinylation, fluorescent labeling, conjugation to carrier proteins, cyclization and incorporation of stable isotopes are routinely done. In addition, the Core supports isoelectric focusing, 2-D gel electrophoresis and digital gel imaging. The Core has been a member of the Association for Biomolecular Resource Facilities since 1995.
Instrumentation and Applications
The CEM LIBERTY1 is a microwave-assisted, single-channel instrument with UV monitor that generates high-purity peptides with fast cycle times. The synthesis scales are 0.05-3mmol. Peptides are synthesized using FMOC chemistry on solid supports or resins. Many modifications are possible, including N and C terminal labeling or capping, side chain modifications, backbone modifications, and stable isotope labeling. Peptides are purified by reverse-phase HPLC on a Beckman System Gold with a UV detector. Peptide masses are verified by MALDI-TOF mass spectral analysis. Synthetic peptides are delivered as dry lyophilized powders. Yield is based on scale and sequence.
Isoelectric Focusing / 2-D Gel Electrophoresis / Electroblotting:
The technique of two-dimensional electrophoresis involves separating proteins in the first dimension according to charge (isoelectric focusing), followed by MW separation in the second dimension by SDS-PAGE. Isoelectric focusing is performed using a Biorad Protean IEF cell. This system uses immobile pH gradients (IPG) gels adhered to a plastic back.
The proteins are then visualized by staining the gel with Coomassie stain, silver stain or fluorescent dyes. This two-dimensional array will produce spots that correspond to single-protein species in the sample. Using this technique, different proteins can be separated and information such as pI, molecular weight and protein abundance can be determined. 2D gels can be electroblotted to PVDF or nitrocellulose membranes for further analysis.
The Licor Odyssey Imaging system uses direct infrared detection that provides accurate quantification, sensitivity and a wide linear range. Two IR channels can be read simultaneously to probe two separate targets or to increase quantification accuracy by using the second channel for normalization. The Odyssesy is suitable for gels, membranes and glass slides.
The GE Healthcare Typhoon Trio is a variable mode imager that can be used for the acquisition of fluorescent or chemiluminescent data. The scanner is capable of imaging gel sandwiches, agarose and polyacrylamide gels, membranes, microplates, and microarrays. Multiple dyes can be analyzed per sample. After acquisition, the images can be quantitated using ImageQuant TL from GE.
Core Policies and Services
The average turnaround time for standard peptide synthesis is two weeks (longer if modification or non-standard amino acids are required). The peptide yield depends on the length and the purity required. The user will be advised if the project does not seem feasible or if low yields are expected.
Training is required before operating the Protean IEF, the LICOR Odyssey or the Typhoon Trio. Online
calendars are maintained for the Protean IEF, Typhoon and Odyssey. Paper calendars are maintained for
the AKTA and the Agilent. Users must sign up in advance to use these instruments.