Quiz 5 Flashcards
What are some ways to quantify protein concentration?
Absorbance at 280nm
Bradford protein assay
Lowry method
BCA assay
Describe SDS-page
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
What is the matrix in an SDS-PAGE made of?
Formed of acrylamide monomers and bidscrylamide crosslinkers
Stacking gel = 4%
Resolving gel = 8-15%
What is the role of glycine and chloride ions in SDS-PAGE?
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
Why does glycine move more slowly through the gel?
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
What are some SDS-PAGE protein standards?
Dual colour Kaleidoscope Duel Xtra All blue WesternC Unstained
Describe how the gel for SDS-PAGE is formed.
Acrylamide monomer is mixed with N,Nā-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
Name some SDS-PAGE protein stains
Flamingo (5hr) Coomassie blue (2.5hr) Silver (1.5hr) Oriole (1.5hr) Spyro ruby (3hr) Stain-free
Describe some gentle methods of cell lysis
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
Describe detergent lysis
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
Describe enzymatic lysis
Suspension of cells in iso-osmotic solutions containing enzymes that digest the cell wall
Usually followed by another disruption method such as sonication
Mammalian
Name some harsher lysis techniques
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)
Describe French press lysis
Application of shear forces by forcing a cell suspension through a small orifice at high pressure
Bacteria, Yeast/algae/fungi
Describe grinding lysis
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
Describe mechanical homogenization lysis
Homogenization with either a handheld device, blenders, or other motorized devices
Best suited for soft tissues
Green plant material, soft tissue
Describe western blotting
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)
Describe what is in the sandwich of western blotting
Clamp Spongy pad Filter paper Membrane Gel Filter paper Spongy pas Clamp
Describe the set up of western blotting tanks
Gel membrane filter sandwich in buffer tank with electrodes on either sides
What is horseradish peroxydase (HRP)?
Horseradish peroxidase is an enzyme that degrades luminol in the presence of peroxides to produce blue light
Describe chemiluminescence
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
