Gel Electrophoresis
native:
mobility = (voltage)(charge)/(mass)
SDS-PAGE: minimizes contribution of charge
IEF:
minimizes contribution of size
Isoelectric Focusing
• separates proteins by isoelectric points
• large pore size of gel and equilibrium
conditions minimize molecular sieving
• native or denaturing conditions possible
Carrier Ampholytes
• mixture of aliphatic amines
+ either carboxylic or
sulfonic acid groups
• generates pH gradient in
electric field
• gradient range depends on
ampholyte pKa values
• proteins migrate to
position = isoelectric point
Preparative IEF (Rotofor)
• polyester screens separate chamber
into 20 compartments
• fractions rapidly harvested following
electrofocusing
IEF Practical Considerations
• gradient range
• low ionic strength for
maximum resolution
• gels: acetone ppt.
• precipitation problems
• include urea, non-ionic
detergents
• heating
• gradient breakdown
Two-Dimensional Gel Electrophoresis
Protein Detection Following Electrophoresis
General Proteins
• organic dyes (eg.,
Coomassie blue)
• R-250 (slow)
• G-250 (fast)
Silver Staining Methods
• diamine (ammonical)
• non-diamine
• photodevelopment
• silver stain
• 10-100X more sensitive
than CB
• fluorescence
• radioactivity
Specific Proteins
• enzyme activity
• antibody/immunoblot
Radiolabeling Proteins
• metabolic
• amino acids
• post-translational
• chemical
• iodination
• alkylating agents
Autoradiography/Fluorography
• electrophoresis of radioactive proteins
• dry gel and expose to X-ray film
• use intensifying screens for high
energy isotopes
• use fluors impregnated in gel for low
and medium energy isotopes
Enhancement of Autoradiographic
Methods for Detection of Radioisotopes
Detection Sensitivity
2
Isotope Energy
Method
(dpm/mm )
direct
2-5
32
125
High ( P, I)
screen
0.5
direct
15-25
35
14
Medium ( S, C)
fluor
2
3
Low ( H)
fluor
10-20
Enhancement shortens exposure times by 7-10 fold
Phosphor Imaging
• filmless autoradiography
• screens contain 'storage-phosphors'
• traps the energy of radioactive emissions
• sensitive to both b-particles and g-rays
• efficiency ~100% for particle striking screen
• scanning the screen with a laser beam
releases the stored energy as light
• ‘fluorescence’ converted into an image file
for display and quantification
• high sensitivity  short exposure times
• range of 5 orders of magnitude
• screens are 'erased' and reused
Quantifying Proteins
• subjective estimates
• scanning densitometry
• excise bands and count
radioactivity
Protein Detection
General Proteins
• Coomassie blue
• silver stains
• fluorescence
• radioactivity
Activity Gels
• carry out electrophoresis
under native conditions
• or remove SDS following
SDS-PAGE
• some proteins refold
• lower SDS and no heat
• replace with non-ionic
detergent
Specific Proteins
• antibody/immunoblot
Protease Activity
• enzyme activity
• protease activity
• co-polymerize PAG with
• redox reactions
protein substrate
• clear zones following
incubation and staining
Redox Reactions and
Tetrazolium Salts
• reduced tetrazolium salts
form insoluble formazan dyes
• eg, nitro-blue tetrazolium (NBT)
• measure dehydrogenases
and other redox reactions
• coupled reactions
• non-redox reactions also
possible
• eg, phosphatase (BCIP)