Gel Electrophoresis
native: mobility = (voltage)(charge)/(mass)SDS-PAGE: minimizes contribution of chargeIEF: 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 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
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
Isotope EnergyDetectionMethod
Sensitivity(dpm/mm2)
direct 2-5High (32P, 125I)
screen 0.5direct 15-25
Medium (35S, 14C)fluor 2
Low (3H) fluor 10-20
Enhancement of Autoradiographic Methods for Detection of Radioisotopes
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 -particles and -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 DetectionGeneral Proteins• Coomassie blue• silver stains• fluorescence• radioactivity
Specific Proteins• antibody/immunoblot• enzyme activity• protease activity• redox reactions
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
Protease Activity • co-polymerize PAG with
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)
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