Protein Purification by Ion Exchange Chromatography
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Protein Purification by Ion Exchange Chromatography
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Column Chromatography Type Protein Separation is based on Ion Exchange Charge Gel Filtration Size Affinity Binding to a specific molecule
Transcript of Protein Purification by Ion Exchange Chromatography
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
