Protein Sequencing
Midwest Analytical, Inc.

Introduction

Midwest Analytical provides N-terminal protein sequencing services to academic and industrial research laboratories. Protein sequencers from Applied Biosystems are used for the automated Edman degradation.

Called sequences are emailed and chromatograms of the phenylthiohydantoin (PTH) amino acids are attached as a PDF file. If requested, database searches using the BLAST programs of the National Center for Biotechnology Information (NCBI) are performed. There is no additional charge for these searches.

Samples

Most proteins are electroblotted to polyvinylidenedifluouride (PVDF) and stained with Coomassie Blue R-250. Please do not use Pierce GelCode Blue Stain or other pre-made stains. These might contain detergents which interfere with some PTH amino acids. If enough protein is available, running three to five lanes is helpful. Multiple bands can be cut if needed. This is especially important with higher molecular weight proteins or lightly stained bands. If the amount of protein is limited (10-20 pmol), a single lane will be needed. It is recommended that the entire membrane be sent with a copy marking the band(s) with molecular weight.

Proteins in solution are passed through a ProSorb PVDF cartridge from Applied Biosystems. This removes buffer salts and detergent, leaving the protein absorbed into the PVDF. The membrane is then loaded for sequencing.

Radiolabelled proteins blotted to PVDF are analyzed for the release of a radioactive amino acid. The cleaved amino acids are collected directly into scintillation vials. After the solvent (n-butyl chloride) has evaporated to dryness, the vials are sent to the customer for counting.

Helpful References

Paul Matsudaira (ed.), A Practical Guide to Protein Purification for Microsequencing, First (1989) and Second (1993) editions, Academic Press, San Diego, CA.

Prices

Minimum Charge (includes first 5 residues) $250.00, each additional residue $30.00
Transfer of solution sample to ProSorb Cartridge $30.00

Company Information

David McCourt
Midwest Analytical, Inc.
11141E South Towne Square
St. Louis, MO 63123

800.481.0790
314.487.8622

Please send samples to:
Midwest Analytical, Inc.
167 Lamp & Lantern Village, #191
Chesterfield, MO 63017

mccourt@mastl.com