Page 246 - The Manga Guide to Biochemistry

4. Conducting Biochemistry Experiments

It was mentioned on page 230 that the functions of proteins can be verified by using bio-
chemical techniques, but what kinds of experiments do biochemists perform?

There are various experimental methods depending on the field of research, and not
each and every one will be presented here. Instead, I will introduce several experimental
methods that I have used myself.

Column Chromatography

Column chromatography is an experimental method for separating substances that have
the same property from a mixture of substances. For example, we can collect just proteins
with certain properties from the liquid extract of American pokeweed, which was introduced
earlier, or from the liquid obtained after a cow thymus was blended in a juicer. Special resins
are packed in a long, narrow tube made of glass or some other material. Some substances
adhere to those resins and are trapped, allowing for the collection of only those substances
that do not adhere.

Various types of chromatography, such as ion exchange chromatography, gel filtration
chromatography, and affinity chromatography, can be used, depending on the type of resin
or target protein. As an example, here we will look at a method for purifying the enzyme
DNA polymerase a from the thymus of a calf.

As shown below, the cells are smashed by grinding up the calf thymus, and a solution
with a high salt (sodium chloride) concentration is used to extract proteins and other mol-
ecules. This extract (the liquid in the flask) passes through the large glass tube (called the
column) in which the ion exchange resin has been packed. The proteins are broadly divided
into those that are trapped in the column and those that pass through the ion exchange
resin.

Roughly “fractionates” the
proteins according to ion
exchange chromatography

Liquified 1) Compounds that pass through
calf thymus the column

Approximately 1.5 M 2) Compounds that are absorbed
by resin in the column

Column Compounds that flow out with DNA polymerase
low salt concentration α is here!

Compounds that flow out with Sample 1
middle salt concentration

Compounds that flow out with
high salt concentration

232 Chapter 5