Gel Electrophoresis native: mobility = (voltage)(charge)/(mass)

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Presentation transcript:

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 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

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 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 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

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)