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Ion Exchange Laboratory

Ion Exchange Laboratory. Today’s Schedule. Pre-lab discussion Ion Exchange and Spectrophotometer Ion exchange experiment. Examples of Column Chromatography. Ion Exchange Gel Filtration Affinity. Ion Exchange Gel Filtration Affinity. Charge Size Conformation.

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Ion Exchange Laboratory

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  1. Ion Exchange Laboratory

  2. Today’s Schedule • Pre-lab discussion • Ion Exchange and Spectrophotometer • Ion exchange experiment

  3. Examples of Column Chromatography • Ion Exchange • Gel Filtration • Affinity

  4. Ion Exchange Gel Filtration Affinity Charge Size Conformation Basis for molecular separation

  5. Components of ion exchange • A charge solid phase or matrix • Liquid phase contains molecules of different charges • Solutions (eluant) of different charges to influence interactions between liquid and solid phases

  6. Solid matrix exchangers • 1. A cation exchanger: • Matrix negative charge • Exchanges cations • 2. An anion exchanger: • Matrix positive charge • Exchanges anions

  7. You will use ion exchange to separate a sample mixture: • Separate dextran blue from cytochrome c

  8. Dextran Blue: large non-ionic polysaccharide (500,000 MW) • Cytochrome c: positively charge protein (12,400 MW) • Which type of solid phase exchangers should you use to separate these two compounds from a mixture?

  9. Cation Exchanger: Cytochrome c is a cation and Solid Matrix has a negative charge

  10. Cytochrome c is bound to column Beads have a negative charge Cytochrome c: positive charge What happens to the dextran blue? How do I remove the cytochrome c from the bead?

  11. Remove cytochrome c with liquid phase of increased ionic strength. Potassium acetate is a positively charged “eluant” Dextran blue eluted earlier Add a potassium acetate (0.5M)

  12. Steps to separate the mixture: • Separate dextran blue from cytochrome c • Add sample mixture • Add 0.01 M KOAc first • Remove (elute) dextran blue • Add 0.5M KOAc next • Remove (elute) cytochrome c

  13. Preparing the column

  14. Add the mixture and elute with potassium acetate

  15. Analyze FractionSpectrophotometer

  16. Calibration Empty Zero transmission Pure water 100% transmittance

  17. Calibration Spectrophotometer Cuvette (requires 4 ml) T A 0 100 plug Set: 100% transmission with cuvette + water Set: 0% transmission without cuvette

  18. Cytochrome c standard curve: • Dilute Standard curve • You must know how to make serial dilutions for Bio 22. • Determine unknown concentration of cytochrome c

  19. Standard Curve • Obtain tube of 10mg/ml of pure cytchrome c • Dilute (as outlined in your lab manual) • Known cytochrome c concentrations • Measure absorbance for known standards • Measure absorbance of unknowns

  20. Each group must have • Sample mixture • (contains 4 mg/ml cytochrome c) • Pure cytochrome c (10 mg/ml)

  21. At bench • Ion exchange column • 0.01 M Potassium acetate • 0.05 M Potassium acetate • Spectrophotometer • Water • Pipets (I’ll review how to use them) • Test tubes and racks should be at your lab bench

  22. Ion exchange Hand in:due date 9/25: beginning of class • Please hand in a neatly presented: 1. Table with absorbance and corresponding concentration values (construct your OWN table.) 2. A graph of your cytochrome c standard curve, be sure to label the axis 3. The concentration of your purified cytochrome c(mg/ml) 4. The volume (ml) of your purified cytochrome c 5. The total amount of cytochrome c purified (mg) (% recovery) Note: If you diluted your purified cytochrome c remember to use the dilution factor in your calculations

  23. Let’s start (I have included some review slides about the spec. and standard curve)

  24. FYI: Instrumentation: Review Spectrophotometer T A 0 100 plug

  25. Calibration Spectrophotometer Cuvette (requires 4 ml) T A 0 100 plug Set: 100% transmission with cuvette + water Set: 0% transmission without cuvette

  26. What is a standard curve? • A graph that allows a quantitative determination known concentration.

  27. Why do we use standard curves? • To obtain quantitative measurements • In clinical settings • Measurement of blood hormones • Measurement of environmental carcinogens • Measurement of drugs • Measurement of antibodies (such as anti-HIV)

  28. Example of Standard CurveStep 1: Data Collection of Known Values Absorbance 260 nm DNA ug/ml 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.18 0.35 0.60 0.70 0.95

  29. Step 2. Standard Curve 1.0 .8 .6 .4 Absorbance (260nm) (dependent variable) .2 0 .2 .4 .6 .8 1.0 0 DNA (ug/ml) (independent variable)

  30. Step 3. Use the curve to calculate unknowns 1.0 .8 Unknowns Prostate Tumor DNA Abs. 0.85 Normal Prostate DNA Abs. 0.40 .6 .4 Absorbance (280nm) (dependent variable) .2 = unknown values X X 0 X .2 .4 .6 .8 1.0 0 DNA (ug/ml) (independent variable)

  31. FYI: Ion Exchange Chromatography • Generally speaking, a protein will bind to a cation exchange resin if the buffer pH is lower than the isoelectric point (pI) of the protein, and will bind to an anion exchange resin if the pH is higher than the pI.

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