TRANSCRIBED BY ; AL-RAISALMILL M. ALAWI
Molecular Tools for Studying GENES AND GENE ACTIVITY ● Instead of a constant current through the gel, this method uses ●
Molecular Separations pulses of current, with relatively long pulses in the forward direction
Gel electrophoresis and shorter pulses in the opposite, or even sideways,direction ●
● It is very often necessary in molecular biology research to separate ● Is valuable for measuring the sizes of DNAs even as large as some
proteins or nucleic acids from each other. of the chromosomes found in yeast.
● For example, we may need to purify a particular enzyme from a Polyacrylamide Gel Electrophoresis ●
crude cellular extract in order to use it or to study its properties. ● Electrophoresis is also often applied to proteins, in which case the
● Gel electrophoresis uses a gel as an anticonvective medium or gel is usually made of polyacrylamide.
sieving medium during electrophoresis, the movement of a charged ● To determine the polypeptide makeup of a complex protein, the
particle in an electric current. experimenter must treat the protein so that the polypeptides, or
●
subunits, will electrophorese independently
●
● This is a horizontal gel made of agarose. The agarose melts at high ● This is usually done by treating the protein with a detergent (sodium
●
temperature, then gels as it cools. dodecyl sulfate, or SDS) to denature the subunits so they no long
● A “comb” is inserted into the molten agarose; after the gel cools, SDS Advantages
the comb is removed, leaving slots, or wells. ● 1. It coats all the polypeptides with negative charges, so they all
● The DNA is then placed in the wells, and an electric current is run electrophorese toward the anode.
through the gel. ● 2. It masks the natural charges of the subunits, so they all
Ion-Ex
● Because the DNA is an acid, it is negatively charged at neutral pH electrophorese according to their molecular masses and not by
●
and electrophoreses, or migrates, toward the positive pole, or their native charges. Small polypeptides fit easily through the pores
anode. 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 Fi
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.
● A photograph of a gel after electrophoresis showing the DNA
● Without net charge, it is no longer drawn toward the anode, or the ●
fragments as bright bands.
cathode, for that matter, so it stops.
● DNA binds to a dye that fluoresces orange under ultraviolet light,
● This step is called isoelectric focusing because it focuses proteins
but the bands appear pink in this photograph.
at their isoelectric points in the gel.
Determining the Size of a Large DNA by Gel Electrophoresis ● The gel is removed from the tube and placed at the top of a slab gel Affinit
● 01. The relationship between the log of a DNA’s size and its for ordinary SDS-PAGE. ●
● electrophoretic mobility deviates strongly from linearity if the ● Now the proteins that have been partially resolved by isoelectric
● DNA is very large. focusing are further resolved according to their sizes by ●
● 02. Double-stranded DNA is a relatively rigid rod—very long SDS-PAGE.
and thin. The longer it is, the more fragile it is. In fact, large
Molecular Tools for Studying GENES AND GENE ACTIVITY ● Instead of a constant current through the gel, this method uses ●
Molecular Separations pulses of current, with relatively long pulses in the forward direction
Gel electrophoresis and shorter pulses in the opposite, or even sideways,direction ●
● It is very often necessary in molecular biology research to separate ● Is valuable for measuring the sizes of DNAs even as large as some
proteins or nucleic acids from each other. of the chromosomes found in yeast.
● For example, we may need to purify a particular enzyme from a Polyacrylamide Gel Electrophoresis ●
crude cellular extract in order to use it or to study its properties. ● Electrophoresis is also often applied to proteins, in which case the
● Gel electrophoresis uses a gel as an anticonvective medium or gel is usually made of polyacrylamide.
sieving medium during electrophoresis, the movement of a charged ● To determine the polypeptide makeup of a complex protein, the
particle in an electric current. experimenter must treat the protein so that the polypeptides, or
●
subunits, will electrophorese independently
●
● This is a horizontal gel made of agarose. The agarose melts at high ● This is usually done by treating the protein with a detergent (sodium
●
temperature, then gels as it cools. dodecyl sulfate, or SDS) to denature the subunits so they no long
● A “comb” is inserted into the molten agarose; after the gel cools, SDS Advantages
the comb is removed, leaving slots, or wells. ● 1. It coats all the polypeptides with negative charges, so they all
● The DNA is then placed in the wells, and an electric current is run electrophorese toward the anode.
through the gel. ● 2. It masks the natural charges of the subunits, so they all
Ion-Ex
● Because the DNA is an acid, it is negatively charged at neutral pH electrophorese according to their molecular masses and not by
●
and electrophoreses, or migrates, toward the positive pole, or their native charges. Small polypeptides fit easily through the pores
anode. 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 Fi
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.
● A photograph of a gel after electrophoresis showing the DNA
● Without net charge, it is no longer drawn toward the anode, or the ●
fragments as bright bands.
cathode, for that matter, so it stops.
● DNA binds to a dye that fluoresces orange under ultraviolet light,
● This step is called isoelectric focusing because it focuses proteins
but the bands appear pink in this photograph.
at their isoelectric points in the gel.
Determining the Size of a Large DNA by Gel Electrophoresis ● The gel is removed from the tube and placed at the top of a slab gel Affinit
● 01. The relationship between the log of a DNA’s size and its for ordinary SDS-PAGE. ●
● electrophoretic mobility deviates strongly from linearity if the ● Now the proteins that have been partially resolved by isoelectric
● DNA is very large. focusing are further resolved according to their sizes by ●
● 02. Double-stranded DNA is a relatively rigid rod—very long SDS-PAGE.
and thin. The longer it is, the more fragile it is. In fact, large
