Gel Filtration

Gel filtration is a chromatographic technique that separates different molecules on the

basis of size. It is commonly used during protein purification to remove unwanted
proteins from the protein being purified. It can also be used to determine the
molecular weight of a protein.
In gel filtration, a dextran, polyacrylamide, or agarose gel is suspended in buffer and
packed in a glass or plastic column. The sample to be analyzed is applied to the top of
the column and is allowed to run down into the gel. A continuous supply of buffer is
then provided at the top of the column, and, as the buffer runs through the column, the
components in the sample are carried down the gel and separated. The buffer is
collected at the bottom of the column in fractions of constant volume (i.e. 1.0 mL),
and all the fractions are analyzed for the presence of the various components in the
sample. The separation of the components is caused by cross-linking in the gel which
creates pores. Small molecules can penetrate the pores and so are slowed down and
retained as they pass down the column. Large molecules cannot penetrate the pores
and so run down the column quickly. Gels with different degrees of cross-linking
(and therefore different sized pores) are commercially available to separate molecules
in different molecular weight ranges. In this experiment, Sephadex G-75 will be
used. This gel is a dextran capable of separating proteins with molecular weights
between 3000 and 70,000.
For a Sephadex column, the total volume, Vt, is equal to the sum of the volume of the
gel matrix, the volume inside the gel matrix, and the volume outside the matrix. The
total volume is also , in most cases, equal to the amount of the buffer required to run a
substance through the column (also known as eluting a substance) when the substance
is small enough to completely penetrate the pores of the gel. Such a substance is said
to be completely included by the gel. For Sephadex G-75, compounds with molecular
weights less than 3000 are completely included. The volume outside the gel matrix is
known as the void volume, Vo. This is the volume required to elute a substance so
large that it cannot penetrate the pores at all. Such a substance is said to be
completely excluded by the gel. For Sephadex G-75, proteins with molecular weights
greater than 70,000 are completely excluded. Compounds with intermediate
molecular sizes that can partially penetrate the pores elute between the void volume
and the total volume, and are said to be partially included by the gel. The volume of
buffer required to elute any given substance is known as the elution volume, Ve, of the
compound. Thus on Sephadex G-75, a protein with a molecular weight of 60,000 will
be less included than a protein with a molecular weight of 30,000. The larger protein
will have a smaller elution volume and run through the column more quickly than the
smaller protein.

Figure 1(Source: Fundamentals of Biochemistry: Life at the Molecular Level by Donald Voet, Judith G. Voet, Charlotte W. Pratt.)

During protein purification, a mixture of many proteins can be subjected to gel

filtration, and all proteins that have molecular weights different from the one being
purified can be separated out. Thus gel filtration is a powerful technique for purifying
a protein. Gel filtration can also be used to determine the molecular weight of a
protein. To do this, several proteins with known molecular weights are run on the
column and their elution volumes determined. If the elution volumes are then plotted
against the log molecular weight of the corresponding proteins, a straight line is
obtained for the separation range of the gel being used. If the elution volume of a
protein of unknown molecular weight is then found, it can be compared to the
calibration curve and the molecular weight determined.
Gel filtration has many advantages as a biochemical technique. It is relatively simple
to perform, and the mild conditions used tend to prevent denaturation of proteins,
unlike some other techniques. The protein that runs off the column can be collected
and used for further analysis, so no protein is consumed in gel filtration. However,
there are also disadvantages as well. The column must be carefully prepared to obtain
optimal separation. Any cracks or discontinuities in the column will interfere. The
size of the sample and the rate of buffer flow must be strictly controlled. If a column
is run several times, each run must be done under the exact same conditions in order
to compare the different runs. finally, some substances stick to Sephadex and do not
elute properly.
In this experiment, a Sephadex column will be prepared. Five substances of known
molecular weight will be used to construct a calibration curve. Then another protein
will be run on the column, its molecular weight determined, and the results compared
to the known size of the protein.

consequently, the large molecules move

more rapidly through the column, and in
this way the mixture can be separated
(fractionated) into its components

the porosity of the gel can be adjusted to

exclude all molecules above a certain size

Sephadex, Sepharose or Sephacryl, which

are fine porous beads, are trade names for
gels that are available commercially in a
broad range of porosities