Ion Exchange Chromatography Ion Exchange Chromatography Anion exchangers

Ion Exchange Chromatography

Ion Exchange Chromatography Anion exchangers They contain immobilized cationic groups that bind to anions. The most common: a matrix with attached diethylaminoethyl (DEAE), DEAE: matrix-CH 2 N+H(CH 2 CH 3) 2 Cation exchangers They contain immobilized anionic groups that bind to cations. e. g. a matrix with carboxymethyl group(CM) CM: matrix- CH 2 – COO-

• The matrix could be cellulose or agarose. • Proteins have negative or positive charges, so they can bind both exchangers.

The binding affinity of a protein depends on: 1. The presence of other ions that compete with the protein for binding to the ion exchanger. 2. the p. H of the solution which influences the net charge of the protein.

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Procedure • The mixture of the proteins is applied to the column. • The column is washed with the buffer. • The proteins with low affinity to the ion exchanger move faster than the proteins with higher affinity. • Proteins that bind tightly can be eluted by applying a buffer with a higher salt concentration or p. H that reduces the affinity.

• In this experiment two proteins( cytochrom c and myoglobin) will be separated by cation exchange chromatography using Sephadex C-25 resin with a 50 m. M sodium phosphate buffer at p. H 8.

• Under these conditions only one protein ( cytochrom) will become attached to the resin. The cytochrom will be attached to the cation exchanger because it is positive at p. H 8 since it's p. I = 10. 4 while the myoglobin's p. I= 7 so its negative (why? ). • To elute the cytochrom which is attached to the resin sodium chloride is added, the positive sodium ions will replace the cytochrom.

• Remove the buffer solution from the top of the resin bed using a Pasteur pipette and then very carefully add the sample. ( the sample is a mixture of myoglobin and cytochrome c). • 3. Wait for a while till the protein mixture enters the resin then open the screw clip. • 4. Immediately add the phosphate buffer (PH 8) and collect 2 ml fractions, collect the fractions until the first colored protein is fully eluted. • .

• 5. Keep the top of the resin covered with buffer at all times. • 6. Remove the buffer from the top of the resin and replace with Na. CL solution and continue collecting fractions until the second colored protein is fully eluted. • 9. Collect approximately 25 fractions. • 7. Read the absorbance at 400 nm using phosphate buffer as blank for the first protein and Nacl for the second protein.

• 8. Record all your results in a table • Draw a graph of the absorbance against the fraction number • Identify the resulting peaks