1. Protein Purification by Ion Exchange Chromatography

2. Column Chromatography
Type
Protein Separation is based on
Ion Exchange
Charge
Gel Filtration
Size
Affinity
Binding to a specific molecule

3. Ion Exchange Chromatography
Uses a solid matrix with either a positive or negative charge
Separation is based on an equilibrium of the molecules adsorbed to the exchanger versus the elution solvent
Changing the ionic strength or pH of the solvent allows separation of molecules with small differences in charge

4. Two Types of Ion Exchangers
A cation exchanger
Bead is negative and adsorbs positively charged molecules
An anion exchanger
Bead is positive and adsorbs negatively charged molecules

5. If You Are Purifying a Positively Charged Molecule…
Which type of ion exchanger would you use?
Cationic
Anionic
What would be the charge on the matrix?
Positive
Negative

6. Basis for Our Ion Exchange Experiment+ + - - - + - + + - - + + - + + +
Sample: negatively charged protein + + + + +
Beads have a positive charge

7. Basis for Our Ion Exchange Experiment- + + + + + + + + + +
Sample: negatively charged protein + + + +
Beads have a positive charge
How can you remove the sample from the bead?

8. Basis for Our Ion Exchange Experiment
Add a salt solution (potassium acetate)
Negatively-charged acetate competes for bead - -
KOAc +- + + - + - + + + + + + +
Sample:Negatively charged protein + + + + -
Eluant: fluid/sample that is removed from column

9. Our Ion Exchange Experiment+ - - - + + - + + + + - - + + - + + + + +
Mixed Sample
Negatively charged protein: GFP Positively charged protein: Cytochrome C + + + + + + +
Beads have a positive charge
GFP= Green Fluorescent Protein, from jellyfish, used to follow gene expression
Cytochrome C= protein involved in electron transport chain

10. Our Ion Exchange Experiment+ - + + + + + + + + + +
Mixed Sample + + + +

11. Our Ion Exchange Experiment
Add 0.01 M KOAc - + - + + - + - + + - + + - + + + +
Mixed Sample + - - + + + +
Cytochrome C elutes from column

12. Our Ion Exchange Experiment-
Add 0.5 M KOAc + + + - + - + + + + + + + +
Mixed Sample + + + + -
GFP elutes from column

13. Guide for Today’s Lab
Control flow of eluant by removing or replacing the cap on the column
Follow directions on pages 79 for
Packing the column
Separating the sample
Green Fluorescent Protein is negatively charged
Cytochrome C (yellow) is positively charged
Quantifying the sample

14. Guide for Today’s Lab Follow directions on page 7 for
Packing the column (Do ALL steps listed in lab manual, only some are summarized here)
Attach column to ring stand (step 1)
Rinse with 0.01M KOAc (step 3)
Pour slurry into column (step 5)
Wash column with additional 0.01M KOAc (steps 6 & 7)
DO NOT LET THE COLUMN RUN DRY

15. Prepare anion exchanger column15

16. Guide for Today’s Lab Follow directions on pages 78 for
Separating the sample
Add sample to top of column bed (steps 1 & 2)
Using 0.01 M KOAc, collect 1 ml fractions until cytochrome C (red dye) has completely eluted (steps 3-5)
Allow remaining 0.01 M KOAc to leave reservoir (step 6)
Using 0.5 M KOAc, collect 1 ml fractions until Green Fluorescent Protein (blue-green dye) has completely eluted (steps 7-10)
Measure the eluted volumes for GFP (step 11)

17. 17

18. Guide for Today’s Lab Follow directions on pages 89 for
Quantifying the sample
Prepare a standard curve for GFP (blue-green dye) (step 1), reading A550 for dilutions from the stock solution (step 1)
Determine the A550 value for each of the fractions containing GFP (step 2)
NOTE: 5 ml samples are required for the spectrophotometer readings. You may either
1. Dilute your fractions at least 1:5 for analysis and then multiply by the dilution factor before extrapolating from the standard curve OR
2. Combine your fractions into one tube, add water if necessary to reach 5 ml and read A550. Correct for the volume when calculating the amount of GFP recovered.

19. Dilutions for Standard Curve Page 8
Read A550THEN Use for next dilution
Read A550THEN Use for next dilution
3 ml
H2O
3 ml
H2O
3 ml
1 mg/ml
3 ml
0.5 mg/ml
6 ml
stock
1 mg/ml
0.5 mg/ml
0.25 mg/ml

20. Spectrophotometer Operation
Set wavelength to 550 nm
Calibration
Empty
Zero transmittance
Pure water
100%
transmittance
Read standards and samples at 550 nm 20