1. Ion Exchange Laboratory

2. Pre-Lab Discussion Outline
Column chromatography
Types and principles
Focus on: ion exchange chromatography
Ion exchange experiment
Sample mixture: GFP and cytochrome c
Pour column
Purification protocol
Spetrophotometer
For your information slides
Spectrophotometer
Standard curve

3. Types of Column Chromatography
Ion Exchange
Gel Filtration
Affinity

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

5. Ion Exchange Chromatography
Separates molecules based on charge
A solid charged cellulose matrix with a charge is used
Solution of different charges are used

6. Types of Ion Exchangers
A cation exchanger
An anion exchanger

7. Today’s Ion Exchange Experiment
Objective: to separate two proteins of different charges from a sample mixture using an ANION EXCHANGER and then determine the concentration of one of these proteins using spectrophotometry.

8. Example of a biomedical application
Let’s say you have a population of cells (tumor + non-tumor) and the tumor cells express an abnormal protein of a different charge (but the same size) as the normal protein. You think this abnormal protein may contribute to tumor growth.
You want to separate these proteins and study them.

9. Obtain sample mixture of two proteins of different charges
Green fluorescent protein(GFP): negatively charged (anionic) chromophores (26.9 kDa)
395 nm (major peak)
(NOTE: due to simulated dye product we will use 550 nm)!
Cytochrome c: positively (cationic) charge protein
(12.4 kD)

10. GFP
238 amino acids
Used to follow gene expression

11. Cytochrome c Highly conserved heme-protein
Associated with inner mitochondrial membrane
Participates in electron transport

12. Prepare anion exchanger column

13. Your colors will be different!

14. Overview of your ion exchange experiment:
Separate GFP from cytochrome c
Add 0.01 M KOAc first
First remove (elute) and discard cytochrome c
Then add 0.5M KOAc next
Remove (elute) and save GFP

15. cytochrome c positively charged (cationic)
Add (0.5 ml) sample mixture (1 mg/ml of starting GFP) to anion exchanger column
Sample mixture:
cytochrome c positively charged (cationic)
and
GFP negatively charged (anionic)
Beads have a positive charge

16. 0.01 M Potassium Acetate
Cytochrome c elutes but GFP remains bound to column
How do you remove GFP from the bead?

17. Now add the: 0.5 M Potassium acetate GFP now elutes Collect sample
Measure volume
Determine concentration

18. Measure the concentration of GFP by spectrophotometry

19. 1st Calibration
Empty
Zero transmission
Pure water
100% transmittance

20. Next: GFP standard curve using serial dilutions:
Volume of GFP
Volume H2O
0.20 mg/ml
1.2 ml of 1 mg/ml stock*
4.8 ml
0.10 mg/ml
3 ml of 0.2 mg/ml
3 ml
0.05 mg/ml
3ml of 0.1 mg/ml
0.025 mg/ml
3 ml of 0.05 mg/ml
0.012 mg/ml
3 ml of .025 mg/ml
0 mg/ml
6 ml
* Stock GFP is 1 mg/ml
BE SURE YOU UNDERSTAND HOW TO PREPARE SERIAL DILUTIONS

21. Finally measure the GFP collected from the column
Obtain absorbance values for your GFP collected from your column
Use your standard curve to convert absorbance to concentration
Sample mixture contained 1 mg/ml of GFP. (remember you added 0.5ml of this solution to your column)

22. Ion exchange worksheet :
Please hand in a neatly presented:
1. Table with absorbance and corresponding concentration values
2. A graph of your GFP standard curve, be sure to label the axis
3. The concentration of your purified GFP (mg/ml)
4. The total amount of GFP purified (mg)
5. The volume (ml) of your collected (purified) GFP
6. The % recovery
= mg GFP purified/total amount GFP (mg) addded x 100
Note: If you diluted your purified GFP remember to use the dilution factor in your calculations

23. Let’s Begin….
These additional slides are to help you review the principles of spectrophotometry and the use of a standard curve.

24. Instrumentation: Review
Spectrophotometer T A
100
plug

25. Calibration Spectrophotometer Cuvette (requires 4 ml) T A plug
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 Curve Step 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 (dependent variable)
Absorbance (260nm) .2 .2 .4 .6 .8
1.0
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
(dependent variable)
Absorbance (280nm) .2
= unknown values X X .2 .4 X .6 .8
1.0
DNA (ug/ml)
(independent variable)

31. FYI: Ion Exchange Chromatography & pH
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.