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-CH2-CH2-

N+H(CH2CH3) 2

Cation exchangers

They contain immobilized anionic groups that bind to cations.

e.g. a matrix with carboxymethyl group(CM)

CM: matrix- CH2 – 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 pH of the solution which influences the net charge of the protein.

•http://higheredbcs.wiley.com/legacy/college/voet/0471214957/animated_figures/ch05/f5-5.html

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 pH 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 50mM sodium phosphate buffer at pH 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 pH 8 since it's pI = 10.4 while the myoglobin's pI= 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.

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• 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 NaCL 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