Exam 1: Protein Electrophoresis and SDS-PAGE

Question 1

SDS-PAGE

Answer 1

Sodium Dodecyl Sulfate

Polycrylamide Gel Electrophoresis

Question 2

Function of SDS

Answer 2

• denatures primary, secondary, and tertiary structures of proteins
• protein unfolds/denatures
  -negative charge will mask all protein charges (uniform negative charge, no matter if positive or negative originally)
• it solubilizes/denatures proteins

Question 3

Proteins migrating through gel

Answer 3

migrate according to size; smaller the peptide, the more rapidly it migrates through gel
- since SDS PAGE give proteins uniform charge, size is determination factor in migration(NOT CHARGE)

Question 4

Gel Electrophoresis Method 1

Answer 4

• native or non-denaturing gel
• separates proteins based on size(mass) and charge

Question 5

Method 2 of Gel Electrophoresis

Answer 5

• Denaturing gel
• separates proteins based on size(mass) after masking intrinsic charge with SDS
• regardless of native charge, a large overall charge is imparted by SDS
• polypeptide will move toward the positive electrode

Question 6

What does B-Mercaptoethanol do?

Answer 6

• breaks S-S bonds, reduces disulfide bonds to separate multi-subunit proteins

Question 7

Ingredients in Sample Buffer/Lamelli Buffer

Answer 7

• SDS
• B-Mercaptoethanol / Dithiothreitol (DDT)
• Bromophenol Blue
• Glyverol
• Tris HCl, pH 6.8

Question 8

Bromophenol Blue

Answer 8

A dye which allows samples to be visualized, for easier loading (you can watch the sample go into the well), and which also provides the gel with a dye front

Question 9

Glycerol

Answer 9

Adds density to the sample, so that it falls to the bottom of the well

Question 10

Tris-HCl, pH 6.8

Answer 10

buffer, which helps keep sample pH and stacking buffer pH roughly equal

Question 11

protein primary structure

Answer 11

sequence of amino acids

Question 12

protein secondary structure

Answer 12

refers to the protein backbone - alpha helix and beta sheets

Question 13

protein tertiary structure

Answer 13

determined by weak bonds and the interaction of the hydrophilic and hydrophobic side chains

Question 14

protein quaternary structure

Answer 14

interaction between multiple polypeptide chains

Question 15

Protein

Answer 15

• enzyme
• amino acids joined by peptide bonds to form polypeptide chains, which may interact with other polypeptides to form multi-sub unit proteins