Lab 11 SDS-PAGE

electrophoresis

powerful method for separating biomolecules in complex mixtures based on the movement of charged particles in an electric field

migration of proteins in an electric field is dependent on…

particle (protein) size, shape, charge (±)

gel electrophoresis

A solid gel matrix is used for electrophoretic
separation of biological molecules

SDS-PAGE

a type of protein electrophoresis with protein migration
through a gel under denaturing conditions

SDS allows..

cell lysis, protein denaturation, evens out charge

Separation results

shape and charge density (constant), size (variable)

PAGE

polyacrylamide gel with acrylamide monomer matrix cross linked with bis-acrylamide to form copolymer

higher bis concentration =

less gaps or pores, smaller pore size, slower protein migration (more polyacrylamide polymers cross-linked)

Polyacrylamide

linear polymer of acrylamide units cross-linked
with bis-acrylamide to create a 3-dimensional matrix

polymerization and cross-linking of acrylamide requires..

catalyst - free radical ions

ex: ammonium persulfate

TEMED

TEMED

stabilizes radical ions long enough for gel polymerization to occur

ammonium persulfate

generates radical ions when dissolved in water

stacking gel

low acrylamide %, lower pH than resolving gel, allows protein sample to concentrate and enter separating gel at the same time, leads to accurate migration, enhanced separation of proteins in sample (sharper beads)

molecular sieving effect

small particles move easier than large, migration speed is inversely proportional to particle size, gel pore size will affect separation range of sample’s proteins

gel pore size depends on…

acrylamide % (4-40%), changing ratio of acrylamide to bis-acrylamide (separation range), typical ratio of acrylamide to bis-acrylamide is 30:1, pH and ionic strength of buffer system affects resolution and separation range

E = V/d

E - field strength

V - voltage

d - distance (cm) between electrodes

Rubisco

most dominant proteins in plant leaves