Protein Purification by Ion Exchange Chromatography
Column Chromatography
Type Protein Separation is based on
Ion Exchange Charge
Gel Filtration SizeAffinity Binding to a specific
molecule
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
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
If You Are Purifying a Positively Charged Molecule…
• Which type of ion exchanger would you use?
1. Cationic2. Anionic
• What would be the charge on the matrix? 1. Positive2. Negative
Basis for Our Ion Exchange Experiment
Beads have a positive charge
Sample: negatively charged protein
+
++ ++ +++++
+
+ ++ +
- --- -
--
Basis for Our Ion Exchange Experiment
Beads have a positive charge
Sample: negatively charged protein
++ ++ +++++
+
+ ++ +
- -
- --
--
How can you remove the sample from the bead?
Basis for Our Ion Exchange Experiment
Sample:Negatively charged protein
++ ++ +++++
+
+ ++ +
- -
-
-
---
Eluant: fluid/sample that is removed from column
Add a salt solution (potassium acetate)Negatively-charged acetate competes for bead-
-
KOAc
-
-+-
-
+
Our Ion Exchange Experiment
Beads have a positive charge
Mixed SampleNegatively charged protein: GFPPositively charged protein: Cytochrome C+
++ ++ +++++
+
+ ++ +
- -- --- +++
++ -
GFP= Green Fluorescent Protein, from jellyfish, used to follow gene expressionCytochrome C= protein involved in electron transport chain
Our Ion Exchange Experiment
Mixed Sample
++ ++ +++++
+
+ ++ +
++ -
- --
-- -+
++
++
Our Ion Exchange Experiment
Mixed Sample
++ ++ +++++
+
+ ++ +
++ -
- --
-- -
+++
+ +Cytochrome Celutes from column
Add 0.01 M KOAc
--
--
-
Our Ion Exchange Experiment
Mixed Sample
++ ++ +++++
+
+ ++ +
++ -
-----
-
GFP elutes from column
-
-
--
--
Add 0.5 M KOAc
-
-- -- -
--
Guide for Today’s Lab
• Control flow of eluant by removing or replacing the cap on the column
• Follow directions on pages 79 for– Packing the column– Separating the sample
• Green Fluorescent Protein is negatively charged• Cytochrome C (yellow) is positively charged
– Quantifying the sample
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
Prepare anion exchanger column
Guide for Today’s Lab
• Follow directions on pages 78 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)
Guide for Today’s Lab
• Follow directions on pages 89 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.
Dilutions for Standard CurvePage 8
stockstock
1 mg/ml 0.25 mg/ml
6 ml3 ml HH22OO
3 ml 1 mg/ml
0.5 mg/ml
3 ml HH22OO
3 ml 0.5 mg/ml
Read A550THEN Use for next dilution
Read A550THEN Use for next dilution
Spectrophotometer Operation
Set wavelengthto 550 nm
Empty Zero
transmittance
Pure water 100% transmittance
Calibration
Read standards and samples at 550 nm