Sds-Page 2018
Sds-Page 2018
Sds-Page 2018
Theoretical Aspects:
Electrophoresis
Electrophoresis is the migration of ions in an electric field, and is an important analytical
technique to separate biological molecules like DNA and proteins. A. Tiselius first used
electrophoresis to separate proteins in 1937.
According to the laws of electrostatic, an ion with charge q in an electric field of strength E,
experiences an electric force,
Felectric = qE
Also, the ion would face frictional force opposing its migration through the solution,
Ffriction = vf
where, v is the velocity of migration of the ion and f is frictional coefficient (depends on the size,
shape and solvation of the ion as well as on the viscosity of the solution).
When the electric field is constant,
Felectric = Ffriction
or, qE = vf
The electrophoretic mobility μ, of the ion, is defined as
μ = v/E = q/f
μ signifies the rate at which the ion would move in a constant electric field and depends on the
sign and magnitude of its net electric charge as well as on its size and shape.
Types of electrophoresis
Depending upon how molecules migrate in electrophoresis during their separation,
electrophoresis can be of two broad types:
2. Zone electrophoresis
When the sample/mixture of molecules to be separated is constrained to move in a solid
support (matrix) like paper or gel, the differently charged molecules migrate according to
their charge, size and shape, and as discrete bands/zones.
Depending on the type of matrix used, it can be of further two types:
(i) Paper electrophoresis
The separation is achieved on a filter paper or cellulose acetate, moistened with a buffer
solution. The ends of the paper are dipped in separate reservoirs of buffer in which
electrodes are placed.
(ii) Gel electrophoresis
It is the most widely used form of electrophoresis for macromolecular separation, and
utilizes gels like agarose and polyacrylamide, which can have variable pore sizes. Gel
electrophoresis therefore adds another dimension to separation, that is, size and shape of
the molecule also affect its mobility in the electric field. The gel electrophoresis can be
horizontal or vertical depending upon the particular apparatuses used; the gels can take
form of slabs/sheets (when horizontally placed, or vertically sandwiched between glass
plates) or columns (when in capillaries). The two most common forms of gel
electrophoresis based on the types of gels used are:
(a) Agarose Gel Electrophoresis (b) Polyacrylamide Gel Electrophoresis
Agarose is a linear polymer of Polyacrylamide is a polymer of straight
alternating D-galactose & 3,6- chains of acrylamide, cross-linked by N,
anhydro-L-galactose. N’-methylenebisacrylamide.
Gelling requires cooling (hydrogen Gelling requires free-radicals & heat
bond formation) (covalent bond formation)
Mostly used to separate medium/ Mostly used to separate proteins & very
large DNA fragments small DNA fragments
Mostly in the form of horizontal Mostly in the form of vertical gel
gel electrophoresis electrophoresis
Range of separation of DNA molecules: Range of separation of proteins:
Agarose conc. Size range of linear Acrylamide conc. (%) Size range of proteins (kD)
(%) DNA fragments (kb)
0.5 30 - 1 5.0 57-212
0.7 12 - 0.8 7.5 36-94
1.0 10 – 0.5 10 20 –80
1.2 7 – 0.4 12 12 - 60
1.5 3 – 0.2 15 10-43
Another useful modification of gel electrophoresis that utilizes two separate gel concentrations
and buffer systems is discontinuous pH or disc electrophoresis:
Stacking/Spacer gel Separating/Resolving gel
Large pore size/ less acrylamide conc. Small pore size/ more acrylamide conc.
pH lower than resolving gel pH higher than stacking gel
Concentrates & stacks the proteins in Resolves/separates/filters proteins
the sample on to the resolving gel according to their size
Placed above resolving gel Placed below stacking gel
The stacking gel thus functions to concentrate the proteins in the samples to aid in their final
separation in the resolving gel. The discontinuous pH system was originally developed in 1964,
independently, by Ornstein and Davis. This system uses the a gel loading buffer (first developed
by Laemmli, and therefore, also called Laemmli buffer) which has glycerol (to add viscosity to the
sample), a buffer identical to that of stacking gel, 0.1% SDS, β-mercaptoethanol (β-ME; to
denature proteins by reducing disulphide bonds), and bromophenol blue (as a tracking dye).
Materials required:
Biological
Cell lysate of Escherichia coli
Chemicals/reagents
30:0.8 Acrylamide:bisacrylamide solution
1.5M Tris-HCl buffer, pH 8.8
0.5M Tris-HCl buffer, pH 6.8
10% APS (made fresh)
10% SDS
TEMED
Electrophoresis/running/tank buffer, pH 8.3 (made fresh)
M. Wt. Composition For 500 mL
Glycine 75.07 190mM
Tris 121.14 25mM
SDS 0.1%
Destaining solution
Composition
Methanol 10%
Glacial acetic acid 10%
Equipment
Vertical gel electrophoresis apparatus with power supply
Micropipettes with microtips, beakers, gel staining dish/box, microcentrifuge tubes, water bath
Procedure:
Assembly of gel plates & casting apparatus
Clean the glass plates, spacers & comb, using distilled water first and then with ethanol.
Three spacers are sandwiched on the edges in between the two gel plates-plain and notched-
on the three sides, leaving the top side of the notched plate.
Large binder clips/clamps are used to hold the plates on the three sides. The three sides of
the sandwiched plates are then sealed by pouring a thin layer of (0.8-1%) agarose/agar in
between the plates on the inner edges.
Mix all the chemicals in the order as given above; APS and TEMED must be added just
before pouring the gel solution.
Pour the gel solution slowly into the assembled gel plates, without introducing any air
bubbles. Fill upto ~ 3/4th of the length of gel plates.
Keep vertically and gently over layer with ~0.5ml of water or butanol. (Oxygen mops up free
radicals and so the gel solutions are usually degassed. For the same reason, in order to avoid
contact with air, gel solution during polymerization is covered with a layer of water.)
Let it polymerize for minimum 30 minutes.
Mix all the chemicals in the order as given above; APS and TEMED must be added just
before pouring the gel solution.
Pour the gel solution slowly over the polymerized resolving gel, after removing water
completely.
Keep vertically and gently insert the comb, leaving at least 0.5cm of stacking gel below the
well of the comb.
Let it polymerize for minimum 30 minutes.
Sample preparation
To the given bacterial cell lysate, add 5X Laemmli buffer (loading dye/buffer) to a final
concentration of 1X.
Boil for 5minutes in a boiling water bath.
Keep on ice till the gel is ready to be loaded.
Loading the samples & electrophoresis
Remove the comb gently after polymerization.
Wash the wells by gently flushing of running buffer, using a syringe. This is done to remove
unpolymerized acrylamide.
Remove the clamps.
Remove the lower spacer carefully without disturbing the gel.
Place the gel plates into the electrophoresis apparatus, tighten the screws.
Pour the running buffer into the lower reservoir (~1/3 rd length of the mini gel tank) and
gently place the electrophoresis apparatus into the gel tank.
Fill the upper reservoir of the tank with running buffer.
Slowly load the sample into the well using a micropipette, avoiding entry of any air bubble.
Attach the electrical leads so that the proteins will migrate towards the positive anode
(red lead). Apply a voltage of 70-100V and allow it to run till bromophenol blue (of the
loading dye) has migrated almost to the lower edge of the gel.
Turn off the electric current and remove the gel plates (with gel sandwiched between them)
from the gel apparatus. Keep it horizontally on a flat surface. Gently pry off one of the glass
plates by inserting a metal spatula in between the plates, avoiding damage to the gel and
breakage of glass plates.
Mark the gel for orientation, maybe by making a small oblique cut on one edge.
There are a number of ways for staining proteins separated in SDS-polyacrylamide gels like
Coomassie blue, Amido black and silver staining. The Coomassie blue binds to arginine, histidine,
and the aromatic amino acids in the proteins, and thus it is highly specific. The ease with which
Coomassie blue is used to stain proteins makes it a widely accepted method for routine
laboratory purposes. The detection of proteins present in very small quantity can be increased
by using silver staining methods, which are more sensitive than Coomassie blue stains.
Precautions:
There shouldn’t be any finger prints, dust particles, or grease marks on the plates.
Entry of air bubbles must be avoided while inserting the comb into the stacking gel.
Prick the lid of the microfuge tube before boiling, to avoid popping it off.
Take care to entry of avoid air bubbles below the lower margin of gel, while placing the
gel apparatus into the electrophoresis tank.