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.
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.
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