I\'ve tried this question but I get confused on b and c. Is this correct? 7. You

Question

I've tried this question but I get confused on b and c. Is this correct?

7. You have a mixture of proteins with the following properties: Protein Molar Mass 45,000 13,400 17,000 69,000 90,000 pl 5.4 10.6 7.0 4.8 5.9 Other factors aside, what order of emergence would you expect from these proteins run on the below methods of separation? (Note: order of elution is what protein elutes first from the column or with gel electrophoresis, what is the order of the proteins from the bottom of the gel to the top) A anion exchange resin at pH 7.0 with a linear salt gradient? a. 5. C, E, A, D b. A gel exclusion column C, A, 0, E c. A SDS polyacrylamide gel electrophoresis A isoelectric-focusing experiment indicate their distribution between the positive (+) and negative (-) ends of the gel. Cathode ()(+) Anode d. 3

Explanation / Answer

Answer:

7. (b) Size Exclusion Chromatography (SEC) is a chromatographic method which separates analytes solely based on their size, where molecules are separated on the basis of their exclusion from pores in the column packing material.

Larger analytes will elute first, while the smaller molecules interact more with the stationary phase and will elute later.

Order of elution: (In order of first to last elution)

i. E = 90 kDa

ii. D = 69 kDa

iii. A = 45 kDa

iv. C = 17 kDa

v. B = 13.4 kDa

(c) When proteins are separated by electrophoresis through a gel matrix, smaller proteins migrate faster due to less resistance from the gel matrix. Other influences on the rate of migration through the gel matrix include the structure and charge of the proteins.

In SDS-PAGE, the use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel largely eliminates the influence of the structure and charge, and proteins are separated solely based on polypeptide chain length.

Order of the protein bands: (In order of maximum migration to minimum migration from the lanes)

i. B = 13.4 kDa (bottom of the gel / maximum migration)

ii. C = 17 kDa

iii. A = 45 kDa

iv. D = 69 kDa

v. E = 90 kDa (Top of the gel / minimum migration)

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