DETECTION AND ANALYSIS OF PROTEINS - A Guide to Methods in the Biomedical Sciences - page 17

several changes of the desired buffer, the buffer within the dialysis tub-
ing will be replaced by the buffer outside of the tube by coming into
equilibrium with the outside buffer. The dialysis tubing can have various
exclusion sizes, such that small molecules below a particular molecular
weight cutoff (1,000 Da, 10,000 Da, etc.) can also be dialyzed out of the
protein mix.
Ultrafiltration is a process by which the volume in which macro-
molecules have been obtained can be concentrated. Ultrafiltration can
be done under pressure or using centrifugal force. The material to be
concentrated is applied in the upper chamber above a filter with the de-
sired molecular weight exclusion so that the protein of interest does not
go through the filter but other small molecules and buffer do. Proteins
mixtures can be concentrated 10 to 100-foid or more using this method.

C. Characterization of primary, secondary, tertiary

and quaternary structures of proteins

Protein sequencing (primary protein structure)

Three different methods were initially developed for direct sequenc-
ing of proteins, These included the Sanger, Dansyl chloride, and
Edman degradation techniques. All three were laborious, required huge
amounts of starting material (grams or more), and also required long
periods of time, often years, to complete the sequence of a short pro-
tein. For the development of one of the methods of protein sequencing
and using it to solve the sequence and structure of insulin, Frederick
Sanger received the first of his two Nobel Prizes in Chemistry in 1958.
He was to receive his second in 1980 for developing what became the
most widely used method for sequencing DNA.
The ability to indirectly deduce the sequence of a protein has come
a long ways since the 1950s and 1960s, and these original methods
are only rarely used today. The most common ways to sequence pro-
teins now is to clone its corresponding cDNA or genomic sequence and
deduce the amino acid sequence from the nucleotide sequence. Us-
ing this method, the genomes of entire species, including man, have
been obtained in a relatively short period of time (see Chapter 2). In
addition, mass spectroscopy is now commonly employed to aid in the
identification and sequencing of proteins.

Mass Spectrometry (MS)

A mass spectrometer is an instrument that can determine the mass of
molecules that have been ionized (electrically charged). MS has become