Protein Analysis Services

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Protein Sequencing

Sequence analysis of purified polypeptides are performed using an Applied Biosystems Model 492HT Protein/Peptide Sequencer equipped with an on-line PTH-amino acid analyzer and data analysis computer. Between 1.0 and 100 pmol of purified peptide is required for sequence analysis.

The chromatographic data from sequence analysis are interpreted by the technician and the laboratory director, and then the best interpretable amino acid sequence is reported to the investigator. Extended amino acid sequences (>10 residues) are searched against the latest available version of the protein sequence databases (including SwissProt and PIR) to identify similarities with known proteins.

Peptide Synthesis

Synthetic peptides containing up to 50 residues are prepared using an Applied Biosystems Model 430a automated peptide synthesizer employing the FASTMoc chemistry protocol. The synthesized peptide will be deprotected and cleaved by trifluoroacetic acid.

Crude peptides will be supplied to investigators as lyophilized solids following deprotection and cleavage from the resin. These peptide will be analyzed by analytical reverse phase HPLC and by quantitative amino acid analysis. If requested by the investigator, amino acid sequence analysis will be performed and charged as stated under Protein Sequencing Cost.

Amino Acid Composition Analysis

Quantitative amino acid composition analysis of hydrolyzed peptides are performed by HPLC following hydrolysis and derivatization with phenylisothiocyanate. The method is used to detect and quantify the common amino acids obtained by acid hydrolysis with a limit of quantitation of approximately 25 pmol. Tryptophan, asparagines, cysteine, and glutamine are not generally determined.

By special request, cysteine can be determined as cysteic acid following performic acid oxidation. The Protein laboratory performs the hydrolysis, derivatization with phenylisothiocyanate, and analysis of the samples by HPLC. The results are reported as moles of each amino acid present in the sample after hydrolysis.

Peptide Mapping

We can do a proteolytic digest of your protein submitted from a SDS-PAGE gel piece or blotted to PVDF membrane. The resulting peptides are separated by HPLC and subsequent amino acid sequencing and/or mass spectrometry analysis of the peaks will be used to determine the internal amino acid sequence of your protein.

Other Services

  • Peptide purification by HPLC
  • HPLC purification of proteins
  • HPLC purification of oligonucleotides and other molecules
  • FPLC chromatography protein purification
  • Open column chromatography
  • Computer analysis of both experimental and theoretical data

Quick Reference

Protein Analysis Lab
Mark Morris
Technician

Phone 336-716-2581
Fax 336-777-3242

jmmorris@wakehealth.edu

Director: Mark O. Lively

Location:
Room 107
Bowman Gray Technical Center
Building 611-13W

Shipping Address:
Attn: Mark Morris / PACL
Dept. Biochem/CSB[BGTC]
Medical Center Blvd.
Winston-Salem, NC 27157

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Last Updated: 03-18-2014
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Disclaimer: The information on this website is for general informational purposes only and SHOULD NOT be relied upon as a substitute for sound professional medical advice, evaluation or care from your physician or other qualified health care provider.