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)