Ion Exchange Laboratory

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Ion Exchange Laboratory
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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

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Types of Column Chromatography

• Ion Exchange
• Gel Filtration
• Affinity

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Basis for separation

• Ion Exchange
• Gel Filtration
• Affinity

• Charge
• Size
• Conformation

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Ion Exchange Chromatography

• Separates molecules based on charge
• A solid charged cellulose matrix with a charge is
  used
• Solution of different charges are used

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Types of Ion Exchangers

• A cation exchanger
• An anion exchanger

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Todays 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.

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Example of a biomedical application

• Lets 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.

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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)

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GFP

• 238 amino acids
• Used to follow gene expression

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Cytochrome c

• Highly conserved heme-protein
• Associated with inner mitochondrial membrane
• Participates in electron transport

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Prepare anion exchanger column
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Your colors will be different!
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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

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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
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0.01 M Potassium Acetate
Cytochrome c elutes but GFP remains bound to
column
How do you remove GFP from the bead?
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Now add the
0.5 M Potassium acetate
GFP now elutes ?Collect sample ?Measure
volume ?Determine concentration
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Measure the concentration of GFPby
spectrophotometry
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1st Calibration
Empty
Zero transmission
Pure water
100 transmittance
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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
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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)

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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
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Lets Begin.
These additional slides are to help you review
the principles of spectrophotometry and the use
of a standard curve.
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Instrumentation Review

• Spectrophotometer

T A
0
100
plug
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Calibration
Cuvette (requires 4 ml)

• Spectrophotometer

T A
0
100
plug
Set 100 transmission with cuvette water Set
0 transmission without cuvette
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What is a standard curve?

• A graph that allows a quantitative determination
  known concentration.

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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)

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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

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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)
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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
.2
.4
.6
.8
1.0
X
0
DNA (ug/ml)
(independent variable)
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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.