For the proteins listed below indicate how you would effectively separate each p
ID: 203001 • Letter: F
Question
For the proteins listed below indicate how you would effectively separate each protein from the other proteins in that mix by answering the questions. Protein name Function pl based on amino acid sequence itongodb bn Size based on amino acid sequence (in kDa) 215 210 7.5 Pro1P Flxip 7.3 120 5.4 Hmmip Cncip Binds to AMP and Ca* Protein kinase, catalyzes the reaction between a protein and ATP to give ADP and a phosphorylated protein Binds to iron (Fe**) Protein phosphatase, catalyzes the reaction that removes a phosphate from a phosphorylated protein Unknown but does bind to Zinc metal ions 35 8.8 Unkp 7.8Explanation / Answer
3a. Pro1P and Flx1p proteins are hardest to seprate becouse there pI value of these protein is near neutral pH. When the protein's pI, equals or near at a neutral pH, the net charge is zero, and the protein is not bound to any exchanger, and therefore,it is hardest to seprate these proteins.
3b. The two techniques that exploit the overall charge of proteins and size are ion-exchange chromatography and Size exclusion chromatography.
Ion exchangers bind charged molecules, and there are essentially only two types of ion exchangers, anion and cation. The net charge of a protein depends on the pH positive at very low pH, negative at high pH, and zero at some specific point in between, termed the isoelectric point (pI). Ion exchangers consist of immobilized charged groups and attract oppositely charged proteins. Protein purification using ion-exchange chromatography has mainly employed positively charged anion exchangers, for the simple reason that the majority of proteins at neutral pH are negatively charged.
In size-exclusion chromotography, the stationary phase is a bunch of beads with pores. Unlike a gel, the beads are not attached to each other, allowing bigger molecules to pass through the spaces between them. Both large and small molecules are slowed down by transient intermolecular bonds to the beads, but the force of gravity on the bigger molecules is greater than on the smaller molecules, so gravity overcomes bead interactions for bigger molecules faster, and bigger molecules elute first in size exclusion chromotography
3c. Disadvantage:
Disadvantage of ion-exchange chromatography:
Along with the benefits, there are certain disadvantages associated with ion exchange, such as iron fouling, calcium sulfate fouling, organic contamination from the resin, adsorption of organic matter, bacterial contamination and chlorine contamination.
Disadvantage of size-exclusion chromotography:
Filtrations must be performed before using the instrument to prevent dust and other particulates from ruining the columns and interfering with the detectors
The molecular masses of most of the chains will be too close for the GPC separation to show anything more than broad peaks