Protein Lab 3 PDF

## Ion Exchange Chromatography ### Introduction Ion exchange chromatography is a separation technique used for purification or analysis of molecules based on their charge. It can be used to separate charged molecules from uncharged ones or molecules of different charges. ### Principle of the meth...

## Ion Exchange Chromatography ### Introduction Ion exchange chromatography is a separation technique used for purification or analysis of molecules based on their charge. It can be used to separate charged molecules from uncharged ones or molecules of different charges. ### Principle of the method Ionizable chemical groups are immobilized on a solid support such as cellulose or agarose. The support, or resin, is usually maintained in a column. Molecules of opposite charge can bind the column by electrostatic interaction while uncharged residues will pass through. Once bound to the column, molecules can be released with salt (NaCl is commonly used, but other salts can be used also). The salt ions compete for interaction for the column, and the molecule of interest is released. Hence the term "ion exchange". Ion-exchange chromatography preserves analyte molecules on the column based on ionic interactions. - **Mobile phase**: buffer, pH and salt concentration - opposite charged solute ions attracted to the stationary phase by electrostatic force. - **Stationary phase**: resin is used to covalently attach anions or cations onto it ### Types of IEC The name of the resin refers to the molecules being exchanged, not the molecule bound to the resin. - **Anion exchangers** - **Cation exchangers** ### Cation exchange chromatography Positively charged molecules are attracted to a negatively charged solid support. Commonly used cation exchange resins are S-resin (sulfate derivatives) and CM resins (carboxylate derived ions): - S-cation exchanger: Resin-CH₂-SO₃^- - CM-cation exchanger: Resin-O-CH₂-COO^- ### Anion exchange chromatography Negatively charged molecules are attracted to a positively charged solid support. Commonly used anion exchange resins are Q-resin (a Quaternary amine) and DEAE resin (DiEthylAmino Ethane): - **Q-anion exchanger**: Resin-CH₂-N⁺(CH₃)₃ - **DEAE-anion exchanger**: Resin-CH₂CH₂-NH₂⁺CH₂CH₃ #### Proteins will bind to an ion exchanger with different affinities. - As the column is washed with buffer, those proteins relatively low affinities for the ion exchange resin will move through the column faster than the proteins that bind to the column. - The greater the binding affinity of a protein for the ion exchange column, the more it will be slowed in eluting off the column. - Proteins can be eluted by changing the elution buffer to one with a higher salt concentration and/or a different pH (stepwise elution or gradient elution). **Cation exchangers bind to proteins with positive charges.** **Anion exchangers bind to proteins with negative charges.** In this experiment, you will separate adenosine 5'-monophosphate (AMP) and adenosine 5'-triphosphate (ATP). These compounds will be separated by chromatography on diethylaminoethyl (DEAE) cellulose. The mixture of compounds will be loaded onto the column, and eluted with a NH4Cl/NH3 gradient. ### Reagents and Materials: - Compound mixture: AMP and ATP - Buffer: 0.25M NH4Cl/NH3, pH 9.0 ### Experimental Procedure 1. Prepare a column of DEAE-cellulose by placing a filter paper at the bottom of a column to serve as a plug. Add a slurry of DEAE-cellulose equilibrated in 0.05M buffer. The final height of DEAE-cellulose in the column should be between 7 to 8 cm. 2. Prepare 10ml solutions of eluting buffer from the stock 0.25M solution. The concentrations should range from 0.05M to 0.25M in increments of 0.05M. 3. Drain the column to just above the top of the resin (do not let resin go dry). Add 1 ml of the compound mixture and allow it to run into the column. 4. Cap the column, and start collecting effluent. 5. Have a test-tube rack ready with 20 numbered test-tubes. Collect ~3 ml of effluent in each tube. 6. Measure the absorbance of each tube at 260 nm. ### Data Analysis 1. Tabulate absorbance of column eluent at 280 nm vs fraction number. 2. Graph your data, plotting absorbance vs fraction number. 3. Draw the structural formulae of the predominant chemical species for the compounds separated in this experiment. lonic charges in the species must be clearly labeled. ### Advantages - It is a non-denaturing technique. It can be used at all stages and scales of purification - An IEX separation can be controlled by changing pH, salt concentration and/or the ion exchange media - It can serve as a concentrating step. A large volume of dilute sample can be applied to a media, and the adsorbed protein subsequently eluted in a smaller volume - It offers high selectivity; it can resolve molecules with small differences in charge. ### Disadvantages - Costly equipment and more expensive chemicals - Turbidity should be below 10ppm.