Electrophoresis Flashcards
A separation process by
applying an electric field
Under the influence of an electric field
dissolved ions present in a solution will migrate at varied rates and directions in a column or a
surface
Ions of opposite charge will migrate in
different directions and become separated on that basis
Ions of like charge, while migrating in the same direction,
become separated due to different migration rate
The rates of migration is depending on:
(a) The strength of the electric field
(b) The net charge on the molecule
(c) The size of the molecule
(d) The viscosity of the medium through which the ions
travel
Gel electrophoresis
Separations are carried out on a thin flat layer (slab) made of a porous semi-solid insoluble material such as agarose or acrylamide
Gel electrophoresis example
DNA gel electrophoresis, SDSPAGE gel electrophoresis,
Capillary electrophoresis (CE)
Separation happened in a buffer filled fused silica capillary that is typically 10 to 100 um in internal diameter and 40 to 100 cm long, extends between two buffer reservoirs that also hold platinum electrodes
SDS is a detergent that
dissociates and unfolds oligomeric proteins
into its subunits
SDS-PAGE
Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis
The SDS binds to the polypeptides to form
complexes with fairly constant charge to mass ratios
SDS is an anionic detergent, meaning that when dissolved
its molecules have a net negative charge within a wide pH range
The negative charges on SDS destroy
most of the complex structure of proteins, and are strongly attracted toward an anode (positively-charged electrode) in an electric field
The electrophoretic migration rate through a gel is therefore determined only by
y the size of the complexes
In the native mode,
the mobility is affected by molecular weight, shape and charge
In denaturing mode
Sample is heated and treated in SDS solution, which denatures and bind to protein, to form a complex that makes the protein to have large negative charge
SDS are attached to the protein in a constant ratio. Protein now have identical charge density
The proteins are now ready to be separated based on the difference in shape
Under these conditions, all the protein particles would migrate at the same rate,
through a matrix of various hole sizes, as exist in the gel
The larger protein will interact with the gel structure, thus travel slower in the gel
Polyacrylamide gels are formed by the reaction of
acrylamide and bis-acrylamide
(N,N’-methylenebisacrylamide) that results in highly cross-linked gel matrix
The gel acts as a
sieve through which the proteins move in response to the electric field
Proteins contain an overall positive or negative charge; this enables
s the movement of a protein molecule towards the isoelectric point at which the molecule has no net charge
By denaturing the proteins and giving them a uniform negative charge,
it is possible to separate them based on the size as they migrate towards the positive
electrode
Smaller proteins migrate more quickly and
thus move further through the gel
large molecules migrate only
a short distance
In order to separate proteins on a gel solely according to size
all of the molecules must have the same charge-to-mass ratio
After separation by electrophoresis,
protein on the gel cannot be seen
and only if staining is performed, the protein is coloured
Molecular weights are determined simultaneously running
marker proteins of known molecular weight
Estimation of molecular mass by
comparing the migration distance to that of a protein with a known mass
Preparing a standard curve
To determine the molecular weight of the
proteins, one must run standard proteins of
known size on the same gel along with the
samples
