Ion Exchange Laboratory
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
Types of Column
Chromatography
• Ion Exchange
• Gel Filtration
• Affinity
Basis for separation
• Ion Exchange
• Gel Filtration
• Affinity
• Charge
• Size
• Conformation
Ion Exchange
Chromatography
• Separates molecules based on charge
• A solid charged cellulose matrix with a charge is used
• Solution of different charges are used
Types of Ion Exchangers
• A cation exchanger
• An anion exchanger
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.
Example of a biomedical
application
• Let’s say you have a population of cells (tumor + nontumor) 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.
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)
GFP
• 238 amino acids
• Used to follow gene expression
Cytochrome c
• Highly conserved heme-protein
• Associated with inner mitochondrial membrane
• Participates in electron transport
Prepare anion exchanger
column
Your colors will be different!
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
Add (0.5 ml) sample mixture (1 mg/ml of starting GFP) to anion
exchanger column
Sample mixture:
cytochrome c positively
charged (cationic)
and
Beads have a positive charge
GFP negatively charged
(anionic)
0.01 M Potassium Acetate
Cytochrome c elutes but GFP remains bound to column
How do you remove GFP from the bead?
Now add the:
0.5 M Potassium acetate
GFP now elutes
Collect sample
Measure volume
Determine concentration
Measure the concentration of GFP
by spectrophotometry
1st Calibration
Empty
Zero transmission
Pure water
100% transmittance
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
3 ml
0.025 mg/ml
3 ml of 0.05 mg/ml
3 ml
0.012 mg/ml
3 ml of .025 mg/ml
3 ml
0 mg/ml
0
6 ml
* Stock GFP is 1 mg/ml
BE SURE YOU UNDERSTAND HOW TO PREPARE SERIAL DILUTIONS
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)
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
Let’s Begin….
These additional slides are to help you review the principles of
spectrophotometry and the use of a standard curve.
Instrumentation: Review
Spectrophotometer
0
100
T
A
plug
Calibration
Cuvette
Spectrophotometer
(requires 4 ml)
0
100
T
A
plug
Set: 100% transmission with cuvette + water
Set: 0% transmission without cuvette
What is a standard curve?
A graph that allows a quantitative
determination known concentration.
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 antiHIV)
Example of Standard Curve
Step 1: Data Collection of Known
Values
DNA ug/ml
0.0
0.2
0.4
0.6
0.8
1.0
Absorbance 260 nm
0.0
0.18
0.35
0.60
0.70
0.95
Step 2. Standard Curve
1.0
Absorbance (260nm)
(dependent variable)
.8
.6
.4
.2
0
0
.2
.4
.6
.8
DNA (ug/ml)
(independent variable)
1.0
Step 3. Use the curve to calculate unknowns
1.0
Absorbance (280nm)
(dependent variable)
.8
Unknowns
.6
Prostate Tumor DNA Abs. 0.85
.4
Normal Prostate DNA Abs. 0.40
.2
X
0
0
X = unknown values
.8
1.0
.4 X .6
DNA (ug/ml)
(independent variable)
.2
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.