1) SDS-PAGE A) HOW DOES IT SEPARATE PROTEINS? B) what does each main component o

ID: 42409 • Letter: 1

Question

1) SDS-PAGE

A) HOW DOES IT SEPARATE PROTEINS?

B) what does each main component of the sample buffer do?

1) SDS

2) REDUCING AGENT

C) what makes proteins migrate toward the bottom of the gel?

2) In-gel digestion

A) what do we use to visualize bands?

b) what is a common contaminent of proteomic samples and what can we do to prevent it?

c) why is trypsin the enzyme of choice for most proteomic experiments?

d) at which sites does trypsin cleave proteins?

e) what is the end product of trypsin digestion called?

f) why is it beneficial to digest proteins for proteomics?( think in terms of a complex sample)

g) how are you able to make protein identifications from the mass spectra?

A) separation is on the basis of relative molecular mass of proteins.

B) 1. SDS is sodium dodecyl sulphate which is anionic detergent and adds negative charge to the protein and unfolds the secondary structure.

2. reducing agent such as mercaptoethanol breaks the disulfide bridges and further unfolds the protein.

C) the negative charge provides by the SDS detergent makes the protein migrate towards the bottom of the gel (positive end).

2) a) coomassie brilliant blue

b) DNA/ RNA molecules are common contaminants.

c) it is used for performing in gel digestion or proetin to produce peptides for mass spectroscopic analysis

d) site of cleavage is carboxyl end of Lysine & arginine.

e) trypsinized product

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