Quiz 5

Question 1

What are some ways to quantify protein concentration?

Answer 1

Absorbance at 280nm
Bradford protein assay
Lowry method
BCA assay

Question 2

Describe SDS-page

Answer 2

Separation according to size
Proteins run through a discontinuous pore size and pH
Proteins saturated with SDS (negatively charged) move towards the positive electrode
Negative pole is always on top

Question 3

What is the matrix in an SDS-PAGE made of?

Answer 3

Formed of acrylamide monomers and bidscrylamide crosslinkers
Stacking gel = 4%
Resolving gel = 8-15%

Question 4

What is the role of glycine and chloride ions in SDS-PAGE?

Answer 4

Glycine moves through the stacking gel much more slowly than chlorine, sandwiching protein between them
Travel through resolving gel as a very tight band becoming more focused

Question 5

Why does glycine move more slowly through the gel?

Answer 5

Can have mixed charges
As it moves from stacking to resolving, it goes from 0 to -1 charge and thus moves more slowly than the negatively charged chloride ions

Question 6

What are some SDS-PAGE protein standards?

Answer 6

Dual colour
Kaleidoscope
Duel Xtra
All blue
WesternC
Unstained

Question 7

Describe how the gel for SDS-PAGE is formed.

Answer 7

Acrylamide monomer is mixed with N,N’-Methylenebisacrylamide cross-linking monomer
Causes cross link between monomers in long chain
Degree of polymerization depends on amount of crosslinkers and monomers
End up with smaller or larger pores

Question 8

Name some SDS-PAGE protein stains

Answer 8

Flamingo (5hr)
Coomassie blue (2.5hr)
Silver (1.5hr)
Oriole (1.5hr)
Spyro ruby (3hr)
Stain-free

Question 9

Describe some gentle methods of cell lysis

Answer 9

Osmotic: suspension of cells in hypotonic solution; cells swell and burst, releasing cellular context (mammalian cell culture)
Freeze-thaw: freezing liquid in nitrogen and subsequent thawing of cells (mammalian)
Detergent
Enzymatic

Question 10

Describe detergent lysis

Answer 10

Suspension of cells in detergent-containing solution to solubilise the cell membrane
This method is usually followed by another disruption method such as sonication
Mammalian

Question 11

Describe enzymatic lysis

Answer 11

Suspension of cells in iso-osmotic solutions containing enzymes that digest the cell wall
Usually followed by another disruption method such as sonication
Mammalian

Question 12

Name some harsher lysis techniques

Answer 12

Sonication: disruption of cell suspension, cooled on ice to avoid heating and subjected to short bursts of ultrasonic waves (bacteria, yeast/algae/fungi, mammalian)
French press
Grinding
Mechanical homogenization
Glass-bead homogenization: application of gentle abrasion by vortex Ing cells with glass beads (algae/fungi/yeast, bacteria, mammalian)

Question 13

Describe French press lysis

Answer 13

Application of shear forces by forcing a cell suspension through a small orifice at high pressure
Bacteria, Yeast/algae/fungi

Question 14

Describe grinding lysis

Answer 14

Breaking cells of solid tissues and microorganisms with a mortar and pestle
Usually mortar is filled with liquid nitrogen and the tissue or cells are ground into a fine powder
Bacteria, yeast/algae/fungi, seeds, green plant material, soft tissue

Question 15

Describe mechanical homogenization lysis

Answer 15

Homogenization with either a handheld device, blenders, or other motorized devices
Best suited for soft tissues
Green plant material, soft tissue

Question 16

Describe western blotting

Answer 16

Transfer of protein onto a membrane
Primary antibody binds to protein on membrane
Enzyme binds to secondary antibody which binds to first antibody
Enzyme substrate connects with enzyme and releases detection signal (chemiluminescent or colourimetric)

Question 17

Describe what is in the sandwich of western blotting

Answer 17

Clamp
Spongy pad
Filter paper
Membrane
Gel
Filter paper
Spongy pas
Clamp

Question 18

Describe the set up of western blotting tanks

Answer 18

Gel membrane filter sandwich in buffer tank with electrodes on either sides

Question 19

What is horseradish peroxydase (HRP)?

Answer 19

Horseradish peroxidase is an enzyme that degrades luminol in the presence of peroxides to produce blue light

Question 20

Describe chemiluminescence

Answer 20

Light emitted by HRP can be picked up on either film or chemidoc system
Linear ranges of signal saturation differ between film and chemidoc
Standard curves with different amounts of pure protein should be run on western blots to determine what this linear range is