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Buffers

Buffers. pH measurement. Voltage  log [H + ] inside /[H + ] outside. Buffers. Buffering capacity Phosphate, carbonate, Tris buffers Good buffers. Electrophoresis. Bible: Gel Electrophoresis of Proteins: Practical Approach, Hammes and Rickwood, IRL Press Principles

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Buffers

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  1. Buffers Biophysical Tools '04 - Electrophoresis

  2. pH measurement Voltage  log [H+]inside/[H+]outside Biophysical Tools '04 - Electrophoresis

  3. Buffers • Buffering capacity • Phosphate, carbonate, Tris buffers • Good buffers Biophysical Tools '04 - Electrophoresis

  4. Electrophoresis • Bible: Gel Electrophoresis of Proteins: Practical Approach, Hammes and Rickwood, IRL Press Principles frictional force = frictional coeff. x velocity qV=vf Electrophoretic mobility U = v/V = q/f = q/(6πηR) Media • paper • cellulose acetate • starch • polyacrylamide • CH2=CH-CO-NH2 acrylamide • CH2=CH-CO-NH-CH2-NH-CO-CH=CH2 bis-acrylamide • polymerization • TEMEDpersulfatex-link • Agarose Biophysical Tools '04 - Electrophoresis

  5. Molecular sieving electrophoretic mobility and sieving logU = logUo - kC Biophysical Tools '04 - Electrophoresis

  6. Experiment Biophysical Tools '04 - Electrophoresis

  7. Stacking gels and Discontinous buffers • Glycine/Cl buffers • +H3NCH2COO-H2NCH2COO-+H+ • Moving boundary • Sample buffer: • Stacking gel: • Running gel: Biophysical Tools '04 - Electrophoresis

  8. Power and Stains • Geometries • vertical: • horizontal: • rod • slab • mini-gels • Power • Voltage for speed  but heating • Detection • dynamic range • Linearity • Dyes: • Coomasie • 0.1-1µg / band • SYPRO Orange • Silver stain • 10-100 ng /band, • Ethidium bromide • Copper • Radioactivity • Immunodetection Biophysical Tools '04 - Electrophoresis

  9. Analysis Biophysical Tools '04 - Electrophoresis

  10. Sample transfer • diffusion • liquify the gels • electrolution • blot transfer • Southern: DNA • Northern: RNA • Western: protein • electroblot: Biophysical Tools '04 - Electrophoresis

  11. SDS-PAGE • Shapiro et al. (1967) improved by Weber and Osborn (1969) • Triple action of SDS • Unfolded protein, coated with uniform charge has mobility determined by size alone. • Distance migrated=a-b*log(MWt) • Gradient gels • Urea gradient gels • Preparative gels Biophysical Tools '04 - Electrophoresis

  12. Agarose gels • shape and flexibility. • linear DNA • circular • Twists • Pulsed Field Gel Electrophoresis • untangle the DNA Biophysical Tools '04 - Electrophoresis

  13. Capillary Electrophoresis • Huge voltage – no current • v. fast Biophysical Tools '04 - Electrophoresis

  14. Isoelectric focusing • Ampholytes • stable pH gradient • Stable pH gradient is achieved by polyampholytes: • Protein migration in a pH gradient Biophysical Tools '04 - Electrophoresis

  15. 2D gels Isoelectric focusing SDS-PAGE Biophysical Tools '04 - Electrophoresis

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