September 2004, Issue 3

The Tufts University Core Facility (TUCF)

The Tufts University Core Facility (TUCF) has been providing reliable biomolecular products and services to Tufts, Tufts-New England Medical Center, and other academic and industry researchers since 1987. Under the direction of Michael Berne, BA, the seven-member staff provides DNA and protein/peptide synthesis, sequencing, and analysis. The TUCF has been self-supporting since it was started with a single protein-sequencing instrument in laboratory space provided by Tufts and the school of medicine's Department of Physiology. With university and department support, Berne expanded the facility to its current scale.

The oligonucleotide (DNA) synthesis service uses two ABI 394s and an Expedite 8909. Turnaround time is often within 24 hours, with oligos being shipped out, dried down and quantified. The DNA sequencing lab consists of two automated ABI 3100 DNA sequencers with a turnaround time of about two days. Results are posted in password-protected files on the core's server, where researchers can download or view both the text of their sequence and the chromatogram.

The core has five ABI 431 peptide synthesizers employing FastMoc Chemistry, capable of synthesizing 5—10 peptides per day depending on their lengths. Turnaround time is usually less than two weeks. The core's ABI 494 protein sequencer is capable of sequencing at low picomole levels, and can handle samples either in solution or blotted onto PVDF (polyvinylidene difluoride) membranes. Turnaround time is usually one week or less. An additional service available upon request is a search of existing data banks for the amino acid sequence of your protein.

Identification of primary peptide and protein sequences (proteomics) is carried out using high performance liquid chromatography and mass spectrometry (HPLC/MS) on a Finnegan LCQ Deca. "Nanospray has become a popular method for analyzing low amounts of sample and, because of its high sensitivity, it is an ideal method for screening a complex mixture of an enzymatic digest of a gel-purified protein with a site-specific protease (i.e. trypsin). By comparison and correlation of tandem mass spectra (MS/MS) of peptides with predicted MS/MS spectra generated from protein and nucleotide sequence databases, we can now check molecular weights, confirm a sequence and characterize post-translational modifications," Berne says.

MALDI-TOF mass spectrometry is used as a quick tool for mass determination and checking sample purity, and requires only fentamoles of sample. The core's Applied Biosystems Voyager-DE PRO can be run in either linear or reflector mode with an accuracy of 0.1% or better. Turnaround time is usually one day.

The TUCF website http://www.tucf.org provides comprehensive information on the facility and its services, including details on sample preparation. Most of the core's services have convenient online order forms.

Core Director Michael Berne can be reached at his TUCF office at (617) 636-2422, or via email at michael.berne@tufts.edu. The TUCF is located on the 8th floor of the Stearns Building in the Tufts School of Medicine M&V complex at 136 Harrison Avenue in Boston.


 

 

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