TASB B ; AL-RAMI M. AL MOLA BIY
Molecular Tools for Studying GENES AND GENE ACTIVITY
Molecular Separations
Gel electrophoresis
● It is very often necessary in molecular biology research to separate
proteins or nucleic acids from each other.
● For example, we may need to purify a particular enzyme from a
crude cellular extract in order to use it or to study its properties.
● Gel electrophoresis uses a gel as an anticonvective medium or
sieving medium during electrophoresis, the movement of a charged
particle in an electric current.
● This is a horizontal gel made of agarose. The agarose melts at high
temperature, then gels as it cools.
● A “comb” is inserted into the molten agarose; after the gel cools,
the comb is removed, leaving slots, or wells.
● The DNA is then placed in the wells, and an electric current is run
through the gel.
● Because the DNA is an acid, it is negatively charged at neutral pH
and electrophoreses, or migrates, toward the positive pole, or
anode.
● A photograph of a gel after electrophoresis showing the DNA
fragments as bright bands.
● DNA binds to a dye that fluoresces orange under ultraviolet light,
but the bands appear pink in this photograph.
Determining the Size of a Large DNA by Gel Electrophoresis
● 01. The relationship between the log of a DNA’s size and its
● electrophoretic mobility deviates strongly from linearity if the
● DNA is very large.
● 02. Double-stranded DNA is a relatively rigid rod—very long
and thin. The longer it is, the more fragile it is. In fact, large
● DNAs break very easily; even seemingly mild manipula tions,
like swirling in a beaker or pipetting, create shearing forces
sufficient to fracture them.
Pulsed-Field Gel Electrophoresis
● A kind of gel electrophoresis that can separate DNA molecules up
to several million base pairs (megabases, Mb) long and maintain a
relatively linear relationship between the log of their sizes and their
mobilities
● Instead of a constant current through the gel, this method uses
pulses of current, with relatively long pulses in the forward direction
and shorter pulses in the opposite, or even sideways,direction
● Is valuable for measuring the sizes of DNAs even as large as some
of the chromosomes found in yeast.
Polyacrylamide Gel Electrophoresis
● Electrophoresis is also often applied to proteins, in which case the
gel is usually made of polyacrylamide.
● To determine the polypeptide makeup of a complex protein, the
experimenter must treat the protein so that the polypeptides, or
subunits, will electrophorese independently
● This is usually done by treating the protein with a detergent (sodium
dodecyl sulfate, or SDS) to denature the subunits so they no long
SDS Advantages
● 1. It coats all the polypeptides with negative charges, so they all
electrophorese toward the anode.
● 2. It masks the natural charges of the subunits, so they all
electrophorese according to their molecular masses and not by
their native charges. Small polypeptides fit easily through the pores
in the gel, so they migrate rapidly. Larger polypeptides migrate
more slowly
Two-Dimensional Gel Electrophoresis
● The mixture of proteins is electrophoresed through a narrow tube
gel containing molecules called ampholytes that set up a pH
gradient from one end of the tube to the other.
● A negatively charged molecule will electrophorese toward the
anode until it reaches its isoelectric point, the pH at which it has no
net charge.
● Without net charge, it is no longer drawn toward the anode, or the
cathode, for that matter, so it stops.
● This step is called isoelectric focusing because it focuses proteins
at their isoelectric points in the gel.
● The gel is removed from the tube and placed at the top of a slab gel
for ordinary SDS-PAGE.
● Now the proteins that have been partially resolved by isoelectric
focusing are further resolved according to their sizes by
SDS-PAGE.
● The investigators grew E. coli cells in the presence or absence of
benzoic acid.
● Then they staine dalysate of the cells grown in the absence of
benzoic acid with the red fluorescent dye Cy3, so the proteins from
that lysate would fluoresce red.
● They stained a lysate of the cells grown in the presence of benzoic
acid with the blue fluorescent dye Cy5, so those proteins would
fluoresce blue. They performed two dimensional gel electrophoresis
is on:
● 1. The proteins from cells grown in the absence of benzoicacid
● 2, On the proteins grown in the presence of benzoic acid
● 3. The proteins that accumulate only in the absence of benzoic acid
fluoresce red, those that accumulate only in the presence of
benzoic acid fluoresce blue, and those that accumulate under both
conditions fluoresce both red and blue, and so appear purple or
black.
Ion-Exchange Chromatography
● Ion-exchange chromatography uses resin to separate substances,
including proteins, according to their charges.
● Positively charged resins like DEAE-Sephadex are used for
anion-exchange chromatography, and negatively charged resins
like phosphocellulose are used for cation-exchange
chromatography
Gel Filtration Chromatography
● Gel filtration chromatography is one method that separates
molecules based on their physical dimensions.
● Gel filtration resins such as Sephadex are porous beads of various
sizes that can be likened to “whiffle balls,” hollow plastic balls with
holes in them.
● Gel filtration chromatography uses columns filled with porous resins
that let in smaller substances, but exclude larger ones. Thus, the
smaller substances are slowed in their journey through the column,
but larger substances travel relatively rapidly through the column.
Affinity Chromatography
● The resin contains a substance (an affinity reagent) to which the
molecule of interest has strong and specific affinity.
● The power of affinity chromatography lies in the specificity of
binding between the affinity reagent on the resin and the molecule
to be purified.
● The molecule of interest binds to a column coupled to the affinity
reagent but all or most other molecules flow through without
binding. Then the molecule of interest can be eluted from the
column with a solution of a substance that disrupts the specific
binding.
