Reference no: EM132525725

Cation-exchange chromatography is a popular technique for separating and purifying proteins. This is very similar to the separation purification of the mixture of amino acids you observed in Prac 3.

You are asked to purify an enzyme with a pI of 6.5 from a mixture of different proteins

You are provided with the following

· A column packed with cation-exchange resin
· Phosphate buffer at pH 2.6
· Citrate buffer at pH 3.4
· Tris-HCl buffer at pH 8.2
· Sodium hydroxide at pH 12.0
· Pipettes and consumables such as test tubes and beakers
· 10 mL of a protein mixture (1 mg/mL) in citrate buffer at pH 3.4

Questions:

(1) Which buffer would you use to equilibrate your column?

(2) Design a purification procedure describing the loading of the sample onto the column, the sequential addition of buffers and the collection of elution fractions. (You must clearly indicate which buffer you would use for each elution step and the number of fractions you are going to collect.

(3) How would you determine which fraction contained your enzyme of interest?

(4) How would you determine the concentration of the enzyme in the fractions assuming that the enzyme was the only protein present in the fractions.