Protein Quantification, SDS-PAGE Gels and ECL Detection

Question 1

Bradford assay

Answer 1

utilizes Coomassie Brilliant Blue G250 (acidic compound)
used to make a standard curve with either BSA or IgG
the pKa shifts with the amount of protein present

Question 2

Anionic detergents

Answer 2

the protein extraction buffer and Laemmli dye contain SDS

Question 3

Reducing agents

Answer 3

the Laemmli dye contains beta-mercaptoethanol

Question 4

SDS-PAGE electrophoresis

Answer 4

need to ensure dissociation of the proteins in to their individual polypeptides
done by: anionic detergents, reducing agents, heat

Question 5

SDS

Answer 5

binds the protein to make them negatively charged

amount binds is proportional to the molecular weight

Question 6

Discontinuous buffer system

Answer 6

the proteins are run using this system

this means that buffer in the reservoirs is a different pH and ionic strength then the buffer than the gel was made with

Question 7

Stacking gel works

Answer 7

the chloride ions (from Tris-HCl pH 6.5) in the sample and the stacking gel form the leading edge
the trailing edge is the glycine (from Tris-glycine pH 8.3) in the running buffer

Question 8

Resolving gel works

Answer 8

the higher pH of the resolving gel ionizes the glycine and moves them through the stacked proteins
the ionized glycine molecules run directly behind the chloride ions