INDM 30020. Protein purification practical – gel filtration.

Gel filtration, or molecular sieving, is a chromatographic technique for the separation of

proteins in a mixture according to their molecular weight. The stationary phase consists of
beads, typically of a dextran-bassed material sold under the trade name of Sephadex, which
contain pores, the size of which determines the range of molecular weights that may be
separated. The proteins enter the pores of the beads and their progression through the column
is retarded: the smaller the protein, the slower it moves through the column. The volume of
mobile phase required to elute a given protein from the column is called the elution volume
(Ve), and can be used to determine the native molecular weight of a protein when compared
with the elution volumes of proteins of known molecular weights. An important
characteristic of a gel filtration column is the void volume, which is the volume between the
beads, and can be determined by passage of a molecule that has a molecular weight that is
much greater than what can enter the beads (Figure).

Figure. Principles of gel filtration. Large molecules are excluded from most of the available column volume
and move rapidly ahead of the solvent front.

In this experiment you will use gel filtration to separate a mixture of blue dextran (molecular
weight ≅ 2,000,000) and potassium dichromate (molecular weight = 294).

PROCEDURE
1. Pouring the column. Pour the slurry of Sephadex G-10 into an empty column (check
that the stopcock is in the closed position) and allow the gel to settle.
2. Open the stopcock and allow the mobile phase to move through the column until it
reaches the top of the gel, then close the stopcock. Do not let the column run dry.
3. Using a micropipette, gently add 100 µ l of blue dextran/potassium dichromate
mixture to the top of the column. Try not to disturb the top of the gel. Open the
stopcock and allow the mixture to enter the gel and close when the top of the mixture
has reached the top of the gel.
4. Add mobile phase to the top of the column using a Pasteur pipette, again avoid
disturbing the top of the gel.
5. Open the stopcock and collect the mobile phase in a test tube. Make sure the column
does not run dry by continually adding mobile phase to the top.
6. When the blue colour has eluted from the column, use another test tube to collect the
yellow fraction. Record the Ve for each of the components of the mixture.

What is the void volume of your column?

 AKTA prime

This is a demonstration of a protein purification apparatus that is used in research labs. It is

composed of pumps, a detector and an automated fraction collector. A variety of columns can
be attached to the pumps depending of the type of chromatography that is to be conducted
(gel-filtration, ion exchange, affinity). A schematic representation is shown in Figure 2. The
column currently attached to the machine is a small gel-filtration column composing of
Sephadex G-10 (the same material that you used in the first part of the practical). In research
labs this column would be used to separate very large molecules, such as proteins, from very
small molecules, such as salt. Thus this column could be used for buffer exchange or
desalting ammonium sulphate fractions, but would not be effective at separating proteins
according to size (a much longer column would be required).

Buffer
Chart recorder
valve

Injector
Pumps Column Detector Fraction
Collector

Waste

Figure 2. Schematic diagram of the components of AKTA prime protein purification system