Purification of Protein

Purified proteins are nowadays required for a wide variety of applications in research, medicine, and biotechnology.

a. Gel filtration chromatography

Also known as molecular sieve chromatography. Separates proteins on the basis of SIZE. Mobile phase is a buffer in which proteins are soluble. Stationary phase is a column of beads containing pores larger than most of the proteins. Small molecules enter pores and elute (exit column) slowly. Large molecules cannot enter the pores and elute quickly. Intermediate sized molecules enter some of the pores and elute between large and small molecules.

Figure 1. Gel filtration chromatography

Figure 2. a plot of protein concentration of each fraction

b. Ion Exchange Chromatography

Proteins separated on basis of CHARGE. Mobile phase is a buffer in which proteins are soluble. Stationary phase is a column of beads carrying + or -charges beads carry counter ions such as Cl- or Na+.

In Cation exchange chromatography, beads carry negative charge. Very positively charged molecules bind strongly to beads and displace counter ions. Negatively charged molecules are repelled from beads and elute quickly. Somewhat positively charged molecules can bind weakly to the beads. Bound compounds (+ charged) are eluted with a gradient of counter ions (buffer containing increasing concentrations of NaCl) or by a change in pH from least to most positive.

In Anion Exchange Chromatography, Beads carry positive (+) charge. Same principles apply as for cation exchange except OPPOSITE. Most positive elute first. Elute from most positively charged to least positively charged. Use for acidic or neutral proteins.

Figure 3. Ion exchange chromatography

Figure 4. a plot of protein concentration of each fraction

Reference:

Koelman J, Roehm KH, Color Atlas Biochemistry. 2nd ed. Marburg: Thieme, 2005.

http://irfanchemist.wordpress.com/2009/04/19/purification-of-protein/

————————————————————————————————

---

Possibly related posts: (automatically generated)

• No Title
• Mechanism of unfolding comes to light!
• Papers and Tools On CBT2.0