Biomaterial Database

High sensitivity amino acid sequence determination. Application to proteins eluted from polyacrylamide gels. 1976

J Bridgen

An automatic solid-phase procedure is described for determining the amino-terminal amino acid sequence of very small quantities of proteins. The sample is covalently attached to an inert support so that mechanical and physical losses during sequencing are eliminated. High sensitivity is achieved by using an initial coupling with high specific activity phenyl [35S]isothiocyanate followed by a longer reaction with the unlabeled reagent. The radioactive phenylthiohydantoins are identified by autoradiography after two-dimensional thin-layer chromatography. Unlabeled phenylthiohydantoin-amino acids are added to each fraction to assist in the identification and to act as carriers, hence reducing absorptive and extractive losses of the small quantities of sample. The method may be used on proteins eluted from polyacrylamide gels containing sodium dodecyl sulfate without removal of the detergent. Sequences of up to 20 residues have been obtained on quantities of protein ranging from 2.5 to 70 pmol. Results from proteins of hitherto unknown sequence are included.

UI	MeSH Term	Description	Entries
D008722	Methods	A series of steps taken in order to conduct research.	Techniques,Methodological Studies,Methodological Study,Procedures,Studies, Methodological,Study, Methodological,Method,Procedure,Technique
D008832	Microchemistry	The development and use of techniques and equipment to study or perform chemical reactions, with small quantities of materials, frequently less than a milligram or a milliliter.
D011506	Proteins	Linear POLYPEPTIDES that are synthesized on RIBOSOMES and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of AMINO ACIDS determines the shape the polypeptide will take, during PROTEIN FOLDING, and the function of the protein.	Gene Products, Protein,Gene Proteins,Protein,Protein Gene Products,Proteins, Gene
D002855	Chromatography, Thin Layer	Chromatography on thin layers of adsorbents rather than in columns. The adsorbent can be alumina, silica gel, silicates, charcoals, or cellulose. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)	Chromatography, Thin-Layer,Thin Layer Chromatography,Chromatographies, Thin Layer,Chromatographies, Thin-Layer,Thin Layer Chromatographies,Thin-Layer Chromatographies,Thin-Layer Chromatography
D004591	Electrophoresis, Polyacrylamide Gel	Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.	Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein