Classical biochemistry Approaches To Studying Proteins Flashcards Preview

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Flashcards in Classical biochemistry Approaches To Studying Proteins Deck (11):

Native proteins

Tertiary structure and interactions preserved


Advantage of native proteins

Can separate proteins of the same molecular weight


Disadvantages of native proteins

- can't interpret MW for gel
- proteins may migrate to either electrode
- difficult to interpret


Denatured proteins

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


Advantage of denatured proteins

Proteins migrate at a rate proportional to their MW


Reducing proteins

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


Non-reducing proteins

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


Structure of page

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


Components of page gel

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


Steps of western blotting

- 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


Advantages of WB

Allows quantification of relative amounts of protein
Allows detection of less abundant proteins