Size exclusion chromatography: 1. List the three types of column chromatography

Question

Size exclusion chromatography: 1. List the three types of column chromatography and explain how each technique separates molecules. 2. Briefly explain how the chromatography used in this lab functions. 3. If the following mix of molecules were purified using size exclusions chromatography, what would be the order in which the molecules pass through the opening in the bottom of the column? Mixture containing: hemoglobin 65,000 daltons; myoglobin, 17,000 daltons; myosin, 180,000 daltons. First molecule to appear: Second molecule to appear: Third molecule to appear: 4. If a size exclusion chromatography column is said to have an exclusion limit of 40,000 Daltons, would hemoglobin (65,000 Daltons) be fractionated or excluded from the column? 5. Would vitamin B12 (1,350 Daltons) be fractionated or excluded from the column? 6. Why did you need to add more buffer after the protein mixture was loaded onto the column?
Size exclusion chromatography: 1. List the three types of column chromatography and explain how each technique separates molecules. 2. Briefly explain how the chromatography used in this lab functions. 3. If the following mix of molecules were purified using size exclusions chromatography, what would be the order in which the molecules pass through the opening in the bottom of the column? Mixture containing: hemoglobin 65,000 daltons; myoglobin, 17,000 daltons; myosin, 180,000 daltons. First molecule to appear: Second molecule to appear: Third molecule to appear: 4. If a size exclusion chromatography column is said to have an exclusion limit of 40,000 Daltons, would hemoglobin (65,000 Daltons) be fractionated or excluded from the column? 5. Would vitamin B12 (1,350 Daltons) be fractionated or excluded from the column? 6. Why did you need to add more buffer after the protein mixture was loaded onto the column?
1. List the three types of column chromatography and explain how each technique separates molecules. 2. Briefly explain how the chromatography used in this lab functions. 3. If the following mix of molecules were purified using size exclusions chromatography, what would be the order in which the molecules pass through the opening in the bottom of the column? Mixture containing: hemoglobin 65,000 daltons; myoglobin, 17,000 daltons; myosin, 180,000 daltons. First molecule to appear: Second molecule to appear: Third molecule to appear: 4. If a size exclusion chromatography column is said to have an exclusion limit of 40,000 Daltons, would hemoglobin (65,000 Daltons) be fractionated or excluded from the column? 5. Would vitamin B12 (1,350 Daltons) be fractionated or excluded from the column? 6. Why did you need to add more buffer after the protein mixture was loaded onto the column?

Explanation / Answer

SOLUTION:
(1) (a) Ion exchange chromatography:

This utilizes the net charge on the protein. As its isoelectric point is 8.75, chymotrypsin will have net positive charge below pH-8.75 and will have net negative charge above pH-8.75. Thumb rule is to use a resin which is 1 to 1.5 pH units below or above pI. Extreme pH changed solutions should be avoided, as they may denature proteins. Both types of resins can be used. If the solution’s pH is 7-8.5 cation exchange resin should be used as protein carries net positive charge. If the solution’s pH is 9-10.5 anion exchange resin should be used as it carries net negative charge. After making the protein bind to the column, washes should be given with the solution of similar pH, to remove contaminants. pH can be altered slightly in order to remove contaminating proteins with near similar proteins of chymotrypsin. Elution can be achieved using buffers of opposite pH. If cation wxchanger was used, elution buffer should have pH above pI and if an anion exchanger was used pH of eluter should be below pI. These will change net charge of chymotrypsin and elute them from the resin. But this technique is not absolute, as there can be many proteins with similar pI, which can contaminate chymotrypsin.

(b) Size exclusion chromatography:

This separates proteins based on their molecular size. Column utilizes packed beads. These beads are not solidly closed like sand. Instead they are porous. So there will be two types of pours. One is pours inside a single bead and another is pours in between beads, which is called void volume. If the protein is big, it cannot enter into the bead and travels only through void volume and elutes faster. If it is small, it’ll be captured into the beads and takes a long path to come down, hence takes more time.

P-30 type of columns can be used to purify proteins of 2.5 to 40 KDa. Chymotrypsin with 25 KDa fits this range. After getting different elutions, they should be checked for chymotrypsin activity. Flow conditions to obtain fraction containing highest chymotrypsin should be saved and used similarly everytime. This technique is also not completely pure, as there can be proteins with similar molecular weights.

However both ion-exchange and size exclusion can be used for getting better purity.

(c) Affinity chromatography:

This is the best technique to get pure chymotrpsin. This utilizes the properties specific to chymotrypsin. Here the columns can be prepared by covalent crosslinking of the molecules, which can specifically bind with chymotrypsin. For example, specific antibodies or specific inhibitors (recombinant chymotrypsin inhibitors) can be used. Non-cleavable substrates which can bind chymotrypsin but cannot be cleaved by it can also be used. After preparing the required column, mixture of proteins should be passed. Extensive washes should be given with similar buffer (For example, PBS), to remove contaminants. Elution can be achieved using change in pH conditions or free unbound inhibitors.

(2)

The Chromatography is the technique which is used for the separation of the mixture.

Chromatography Technique works as, basically on the 2 Phases mainly the mobile phase and the stationary phase. The mixture is passed through the column , the sample is passed alone with the mobile phase , if the sample has the affinity with the mobile phase then the it will elute first , and if the sample has the affinity towards the stationary phase then that will take more time to elute.

(3)

Chromatographic separation of biomolecules based on differences in molecular weight

Molecules that are larger than the pores of the gel beads in the chromatography column pass quickly around the beads, while the small molecular weight proteins pass through these porous beads taking longer route hence elute out at last.

Order of elution

1. Myoglobin

2. Hemoglobin

3. Myosin

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