Lecture 16

Question 1

Size Exclusion Chromatography

Answer 1

larger moves faster, smaller moves slower

Question 2

Ion Exchange Chromatography

Answer 2

oppositely charged beads stick to column, everything else flows through

Question 3

Affinity Chromatography

Answer 3

Specific trap for protein of interest attached to stationary phase; high purity can be achieved

Question 4

Order of specificity of different chromatography methods

Answer 4

• affinity
• ion exchange
• size exclusion

Question 5

electrophoresis

Answer 5

migration of ions in an electric field

Question 6

In electrophoresis, velocity is directly proportional to ______ and inversely proportional to_________.

Answer 6

charge, size and shape

Question 7

A gel is used to…

Answer 7

slow down movement, prevent diffusion of proteins, and create separation

Question 8

SDS-PAGE

Answer 8

sodium dodecyl sulfate polyacrylamide gel electrophoresis

Question 9

SDS

Answer 9

a detergent that will denature proteins and give them a negative charge

Question 10

How much SDS for 1g protein?

Answer 10

~1.4g

Question 11

acrylamide

Answer 11

acrylic amide; a plastic monomer used industrially that can be chained together

Question 12

monomers

Answer 12

toxic, carcinogenic and teratogenic, but safe when linked to gel

Question 13

SDS-PAGE procedure

Answer 13

• protein samples boiled for 5mins in 5xloading buffer
• gel is cast by mixing acrylamide, bisacrylamide, APS, TEMED, SDS, and buffer
• gel placed vertically in electrophoresis apparatus, tanks are filled with buffer
• samples loaded into wells (ladder important to determine sizes)
• power turned on until blue dye reaches bottom

Question 14

Visualizing gel

Answer 14

-coomassie blue, silver nitrate (complicated and time consuming), fluorescent dye (require specialized equipment)