1. 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

2. 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

3. Preparative IEF (Rotofor)
polyester screens separate chamber into 20 compartments
fractions rapidly harvested following electrofocusing

4. IEF Practical Considerations
gradient range
low ionic strength for maximum resolution
gels: acetone ppt.
precipitation problems
include urea, non-ionic detergents
heating
gradient breakdown

5. Two-Dimensional Gel Electrophoresis

7. Protein Detection Following Electrophoresis
General Proteins
organic dyes (eg., Coomassie blue)
R-250 (slow)
G-250 (fast)
silver stain
10-100X more sensitive than CB
fluorescence
radioactivity
Specific Proteins
enzyme activity
antibody/immunoblot
Silver Staining Methods
diamine (ammonical)
non-diamine
photodevelopment
Radiolabeling Proteins
metabolic
amino acids
post-translational
chemical
iodination
alkylating agents

8. 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

9. Enhancement of Autoradiographic Methods for Detection of Radioisotopes
Enhancement shortens exposure times by 7-10 fold

10. 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

11. Quantifying Proteins subjective estimates scanning densitometry
excise bands and count radioactivity

12. Protein Detection 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
General Proteins
Coomassie blue
silver stains
fluorescence
radioactivity
Specific Proteins
antibody/immunoblot
enzyme activity
protease activity
redox reactions
Protease Activity
co-polymerize PAG with protein substrate
clear zones following incubation and staining

14. 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)