Physical Methods to

Characterize Proteins
Physical properties of key interest

Molecular weight
Oligomerization state

Structure

Interactors
 Transport Processes

1. Transport in an electric field (electrophoresis)

Requires a matrix (either agarose or polyacrylamide) to
minimize convective effects due to heating and Brownian
motion. Mobility is also affected by partioning with the
stationary phase (matrix). Provides relative molecular weights
determined by comparison to standards.

2. Transport in a gravitational field (centrifugation)

Requires a centrifuge to generate the large gravitational
forces required for protein transport. Provides an
absolute molecular weight and informs on the
oligomerization state for stable complexes.

3. Transport by partitioning between mobile and

stationary phases (gel exclusion chromatography)
Requires a suitably-sized partitioning matrix for the solid
phase. Provides relative molecular weights determined by
comparison to standards.
 Gel Electrophoresis

u = A(q/f)
Electrophoretic mobility (u) is proportional to its net charge (q), specifically the
ratio of its net surface charge to accessible surface area, and inversely
proportional to its frictional coefficient (f), a function of solvent viscosity and
protein geometry. The constant of proportionality (A) is unique to each protein.

Mobility can be measured to the cathode
or anode depending on protein charge
and pH.
Free radicals are provided by ammonium persulfate.
TEMED (tetramethylenediamine) is included to stabilize
the free radicals resulting from decomposition of the
ammonium persulfate. Ratio of acrylamide to
bisacrylamide is held constant. Pore size is expressed
as percent (w/v) acrylamide and designated %T.
 Disc (Discontinuous) Gel Electrophoresis

Changes in electrophoretic mobility in

the focusing zone

Leading anion: Cl-

Trailing ion: glycine
 SDS PAGE

SDS: Sodium Dodecyl Sulfate (Lauryl sulfate)

SDS binds protein with a constant ratio of 1.4

gm SDS/gm protein.

Calibration plot for a series of molecular

weight standards vs. %T
 Gel Filtration/Size Exclusion Chromatography
Comparison of size resolution by gel Calibration plots for molecular weight
filtration (left) and PAGE (right) standards resolved on different media

Band spreading as a function of

elution position
 Mass Spectrometry

Ionization methods

Matrix-assisted Laser desorption/ionization (MALDI)

Basic mass spectrometer design

Electrospray ionization (ESI)

Detection methods
Time of Flight (TOF)- Accuracy to 0.1%
Quadrupole mass analyzers- Accuracy to 0.01%
FT ion cyclotron- Accuracy to 0.001%
 Types of MS Data

Mass determination of intact proteins

Tandem mass spectrometry analysis by collision-

induced dissociation (CID)

Tandem mass spectrometry

SILAC-Stable Isotope Labeling by Amino Acids