Classical biochemistry Approaches To Studying Proteins

Question 0

Advantage of native proteins

Answer 0

Can separate proteins of the same molecular weight

Question 1

Native proteins

Answer 1

Tertiary structure and interactions preserved

Question 2

Disadvantages of native proteins

Answer 2

• can’t interpret MW for gel
• proteins may migrate to either electrode
• difficult to interpret

Question 3

Denatured proteins

Answer 3

SDS imparts overall negative charge so all proteins agave equal mass-to-charge ratio and rod like structure

Question 4

Advantage of denatured proteins

Answer 4

Proteins migrate at a rate proportional to their MW

Question 5

Reducing proteins

Answer 5

Reducing agents break disulfide bonds so migration not affected by tertiary/quaternary structure

Question 6

Non-reducing proteins

Answer 6

Disulfide bonds left in tact
So can help elucidate tertiary or quaternary structure of proteins
Can’t gauge MW because structure may retard migration

Question 7

Structure of page

Answer 7

Poly acrylamide = polymer of acrylamide monomers
Polymerize into long chains (initiated by APS and TEMED)
Bis acrylamide forms cross-links

Question 8

Components of page gel

Answer 8

Stacking gel
- large pore, tris-Cl, pH 6.8, proteins conc in single band
Resolving gel
- small pore, tris-Cl. PH 8.8 , proteins separated according to size

Question 9

Steps of western blotting

Answer 9

• separate proteins by page
• transfer proteins from gel to membrane
• block membrane
• incubate with primary ab
• incubate with secondary ab conjugated to enzyme
• incubate with enzyme substrate

Question 10

Advantages of WB

Answer 10

Allows quantification of relative amounts of protein

Allows detection of less abundant proteins