PROTEIN/PEPTIDE SEQUENCER AND SYNTHESIZER

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We are requesting purchase of an Integrated Micro-sequencing System (Model PI 2090E, Porton Instruments, Inc.) and a Peptide Synthesizer (Model 9050, Milligen/Biosearch). The choice of these instruments has been carefully researched and reappraised since our original application of March, 1989. We are convinced, based upon data presently available, that they will best serve the needs of our user group of four connective tissue researchers. Most of the peptide sequencing and synthesis tasks for major and minor users alike relate to questions of structure and interactions of connective tissue elements (eg. proteoglycans and collagens). One of the particular research strengths of UAB is in the connective field. Although connective tissue workers are allied with different departments, many need and will benefit from a peptide/sequencing facility targeted to their problems. We predict that 80% of the operating time of the sequencer will be required for the major users' projects. Many of the peptides presented will be ready-immobilized through electro-blotting to PVDF membranes. The sequencer is suited to sequencing samples in this form and at the required sensitivity (eg. 5 microgram of protein, MW 50 KDa, which is 0.1 nmole/cycle). Changes in the sequencing protocol will be necessary to identify glycosylation sites, which we'll frequently need to do. The selected instrument has the necessary flexibility to permit this. We expect the major users to require about 75% of the operating time of the synthesizer. We have decided to use FMOC chemistry, which has several advantages. For example, there is no need for reagents like HF. And techniques have been developed where synthesized peptides can be coupled in series. The peptide synthesizer selected is, in our opinion, the most versatile and economic instrument on the market.