Molecular Weight
Determination
SDS-PAGE
Electrophoresis
• Movement of particles by an electric force.
• Gel is the matrix perfused by buffer salts
– Matrix will
• reduce effects of diffusion
• stabilize the system against turbulence (thermal agitation)
• Introduce sieving action that allows separation based on molecular size
– Buffer salts will
• prevent sample interactions
• create more conductivity
• generates heat
– Mobility = applied voltage x net charge on the molecule /
function of molecule (shape, size and other physical matter)
– Force: Total electric field is the effective charge and field strength
– Opposing force is friction and pore size
– When charge/mass is constant then large molecules move slower than
small molecules.
Electrophoresis
• Two other factors are important
– Ionic strength of surrounding solution
• Buffer ions –
– high ionic strength reduces effective charge of sample and
temperature will increase;
– low ionic strength slows the movement but has sharper peaks.
• Best is Tris borate buffer – only few ions are free and large
size ions reduce excessive heat generation
– Temperature of the gel
• Effects the viscosity of the gel
• Need to run at constant power
• SDS PAGE maintain high temperature to prevent
renaturation
Electrophoresis
• Electric parameters:
Power = I2R
I = current R= resistance
• Buffer ions: cations move towards
negative electrode in upper chamber and
anions move towards positive electrode in
the lower chamber.
• Apparatus and buffers provide constant
resistance.
Type of electrophoresis
• Solution: Solutes dissolved in a solvent and
electric force is applied.
• Zonal: Aqueous ionic solution is carried in solid
matrix.
– Paper
– cellulose acetate and cellulose nitrate
– Gels
• Starch gels – organelles
• Agarose DNA
• Polyacrylamide – proteins and nucleic acids
Polymerization
• Polyacrylamide gels are formed by the
polymerization of acrylamide in aqueous solution
in the presence of small amounts of a
bifunctional crosslinker, usually bisacrylamide
(bis).
• The copolymerization of acrylamide with
bisacrylamide produces a mesh-like network in
three dimensions, consisting of acrylamide
chains with interconnections formed from the
bisacrylamide.
Polyacrylamide gels
• T -the total percentage concentration (w/v) of
monomer (acrylamide plus crosslinker) in the
gel.
• C - the percentage of the total monomer
represented by the crosslinker.
• For example, an 8%, 19:1
(acrylamide/bisacrylamide) gel would have a T
value of 8% and a C value of 5%.
Polymerization
• The polymerization of acrylamide and
bisacrylamide occurs via a free-radical
mechanism after adding a catalyst.
• ammonium persulfate initiates free
radicals and
• tertiary aliphatic amine N,N,N',N'tetramethylethylenediamine (TEMED) is a
catalyst for free radical propagation
Polymerization
• The presence of excess oxygen will inhibit
the polymerization elongation process and
can lead to shorter average chain length.
• Deaeration under vacuum with a magnetic
stirrer is needed prior to addition of
initiators.
Advantages of Polyacrylamide Gels
• Small pore size.
• By changing the concentration of the
monomer and the cross-linker, the pore
size can be varied in a reproducible
manner.
• It is synthetic so no batch to batch
differences.
SDS-PAGE
• SDS (Sodium dodecyl sulfate) – PAGE (Poly Acrylamide
Gel Electrophoresis) separation of proteins is based on
molecular weight.
• In their native form, proteins fold into a variety of shapes,
some compact, some elongated. The rate of migration of
native proteins through a sieving medium is therefore
more a reflection of their relative compactness, and less
an accurate measure of molecular weight.
• Denaturing the proteins nullifies structural effects on
mobility, allowing separation on a true charge/mass ratio
basis. It also separates subunits in multimeric proteins,
allowing analysis of large, complex aggregates.
SDS
• SDS is amphiphatic surfactant, an anionic
detergent (pH above 7)
• Hydrophobic tail binds to protein backbone
• Polar heads denature ionic bonds
• Uniform coating leading to charge/mass
ratio consistent between proteins. 1.4g
SDS/gram of protein is needed.
SDS-PAGE
• 2-mercaptoethanol – dissociates any
disulfide bonds
• SDS and urea disrupts ionic, hydrogen
and hydrophobic bonds.
• Overall – proteins have negative charge
and disrupted secondary and tertiary
charges.
SDS-PAGE
• Protein now have negative charge. When
electric field is applied they move towards
cathode.
• Larger the size of the protein, the more it
is retarded by contact with the meshwork
of acrylamide in the gel, so the smallest
peptides will move farther in the gel and
the larger molecules move progressively
shorter distances
Discontinuous gel Electrophoresis
• Two layers of different concentrations of gel and
pH
– Stacking low concentration and low pH
– Separating gel – higher concentration of gel and high
pH
• Concentrate proteins in narrow band in stacking
gel before traveling through separation gel for
greater resolution of bands
• Made up of stacking gel pH 6.8; separation gel,
pH 8.8 and the tank .
Discontinuous Gels
• Stacking gel has low concentration of gel, so ions move
freely under applied voltage.
– Glycine and chloride ions are used
• Chloride ions move faster (leading ions) than glycine (trailing ions)
and create steep voltage gradient. Glycine ions are pulled due to
the charge gradient and the two ions move at same speed. The
proteins with intermediate mobility are carried along becoming
stacked into very thin, distinctive layers in order of mobility.
• Resolving gel has high concentration of gel and high pH:
– When the interface between the stacking and separation gel is
reached by moving, the pH changes abruptly.
– Glycine moves quickly and unstacks the proteins.
– The movement of proteins is influenced by sieving capacity of
the gel and its molecular weight because pH and the voltage are
constant.
SDS-PAGE
• After electrophoresis, the gel is treated
with trichloroacetic acid solution
– makes the proteins insoluble and
– prevent them from diffusing during staining
procedure
SDS-PAGE
• Staining to make invisible proteins visible.
• Comassie blue in methanol-acetic acid
Procedure
•
•
•
•
Prepare Tank buffer
2x treatment buffer
Staining solutions
Destaining solutions I and II
Protein Samples
• Dissolve protein sample with 2x treatment
buffer (protein conc. 50 µg). Boil for 5min
to denature.
• Standards:
• Lactalbumin; trypsin inhibitor; trypsinogen;
carbonic anhydrase; albumin egg; albumin
bovine
SDS-PAGE
• Preparing standard curve
– Rf = distance traveled by peptide / distance
traveled by tracking gel
• Plot a standard curve of log molecular
weight Vs Rf