Experimental approach (total 25 p) 1-You are to fractionate a protein with Pi of

Question

Experimental approach (total 25 p) 1-You are to fractionate a protein with Pi of 7.1 in a phosphate buffer ( pH: 5.0), using ion exchange column chromatography a. What type of resin (stationary phase) you have to choose. (negative or positive functional group) or how you will make it b. List the major materials (type of resin) and explain how you make your stationary phase. c. What would be the pH of your elution buffer a. Hints The general technical steps of Colum chromatograph are similar to what you have done Protein in a buffer with the pH lower than its Pi will carry a positive net charge Protein in a buffer with the pH greater than its p will carry a negative net charge Resin Type of Intorent the pl of the aoveeplohe condiion of interest

Explanation / Answer

Background

As we know that isoelectric point is the pH at which a particular protein molecule carries NO net electrical charge or is electrically neutral in the statistical mean, (proteins usually exists in zwitterion form).  pH=pi (isoelectric point)

What is given here is

Protein pi=7.1, and phosphate buffer mobile phase pH=5.0

Which means, mobile phase pH < pi , So the resultant proteins side chains exist with positively charged ions (Cations). (Review Above Point no: 2)

In ion exchange chromatography,

1A) Answer

Stationary phase resin should have anions attached in order to bind, retain or separate proteins with positively charged proteins (Cations).

1B) Answer

Stationary Phase Preparation:

Preparation process includes carefully loading of required porous resin or silica materials into a steel tube of different diameter (10 microns, 5 microns). The resultant bounded silica should be packed uniformly which can withstand certain pressures that packing remains unaltered.

Materials required

1C) Answer:

As per the above conditions to purify the final protein, the pH of mobile phase (or elution buffer) should me maintain lower than pi of proteins. In this case, we can continue the experiment with the same buffer solution having a pH of 5.0

Anion Exchange Resin Cation Exchange Resin 1 Quaternized polyethyleneimine (mixed amine) 1. Sodium polystyrene sulfonate 2 Polyethyleneimine (mixed amine) 2. Patiromer 3 Fully quaternized amine 4 Dimethylaminopropyl

Related Questions

Navigate

• Browse (All)
• Browse E
• Subjects

---

---

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.