Protein Purification by Ion Exchange Chromatography

Column Chromatography Type Protein Separation is based on Ion Exchange Charge Gel Filtration Size Affinity 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? Cationic Anionic What would be the charge on the matrix? Positive Negative

Basis for Our Ion Exchange Experiment + + - - - + - + + - - + + - + + + Sample: negatively charged protein + + + + + Beads have a positive charge

Basis for Our Ion Exchange Experiment - + + + + + + + + + + Sample: negatively charged protein + + + + Beads have a positive charge How can you remove the sample from the bead?

Basis for Our Ion Exchange Experiment Add a salt solution (potassium acetate) Negatively-charged acetate competes for bead - - KOAc +- + + - + - + + + + + + + Sample:Negatively charged protein + + + + - Eluant: fluid/sample that is removed from column

Our Ion Exchange Experiment + - - - + + - + + + + - - + + - + + + + + Mixed Sample Negatively charged protein: GFP Positively charged protein: Cytochrome C + + + + + + + Beads have a positive charge GFP= Green Fluorescent Protein, from jellyfish, used to follow gene expression Cytochrome C= protein involved in electron transport chain

Our Ion Exchange Experiment + - + + + + + + + + + + Mixed Sample + + + +

Our Ion Exchange Experiment Add 0.01 M KOAc - + - + + - + - + + - + + - + + + + Mixed Sample + - - + + + + Cytochrome C elutes from column

Our Ion Exchange Experiment - Add 0.5 M KOAc + + + - + - + + + + + + + + Mixed Sample + + + + - GFP elutes from column

Guide for Today’s Lab Control flow of eluant by removing or replacing the cap on the column Follow directions on pages 79 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 15

Guide for Today’s Lab Follow directions on pages 78 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)

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Guide for Today’s Lab Follow directions on pages 89 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 Curve Page 8 Read A550THEN Use for next dilution Read A550THEN Use for next dilution 3 ml H2O 3 ml H2O 3 ml 1 mg/ml 3 ml 0.5 mg/ml 6 ml stock 1 mg/ml 0.5 mg/ml 0.25 mg/ml

Spectrophotometer Operation Set wavelength to 550 nm Calibration Empty Zero transmittance Pure water 100% transmittance Read standards and samples at 550 nm 20