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