Unit 2 Flashcards
(18 cards)
1
Q
Green Fluorescent Protein (GFP)
A
- Naturally found protein from jellyfish
- Emits green fluorescence when exposed to light of the exciting wavelength
- When linked to protein of interest, GFP can be used to follow localisation & movement of protein of interest in live cells
1
Q
Tissue culture
A
- Cells can be isolated from tissues
- Disrupt cell-cell contacts with a protease, or EDTA which chelates Ca2+
- Plate cells in a plastic dish. Some cells (non-adherent cells) require a layer of collagen to adhere to the plastic, while others adhere naturally (adherent cells)
- If mixed pop. of cells, Fluorescence-activated cell sorting (FACS) can be used to separate them
2
Q
FACS
A
- An antibody-recognising a protein facing the outside surface of a cell is coupled to a fluorescent dye and suspended in fluid
- Droplets with single cells pass by a laser to excite the fluorescent dye. If the detector registers fluorescence, the droplet is immediately negatively charged. Otherwise, the drops are not charged.
- The droplets are then deflected in an electric field and collected. They are then put into culture on plastic dishes.
3
Q
Confluency
A
- % of surface area of a dish a cell occupies
- When at 100%, they must be passaged (to a new dish)
4
Q
Senescent
A
- When cells no longer express telomerase, & stop dividing
- Adding DNA that express telomerase immortalises them. Known as cell line
- Some cancer cells can grow indefinitely & are referred to as transformed cell line
5
Q
Subcellular fractionation
A
- Breaking a cell into its functioning organelles
1. Disruption of plasma membrane under conditions that don’t destroy organelles & placed in isotonic solution of equal pH (~7.5). Done by:
a.) Blender
b.) Sonication (exposure to high-frequency sound)
c.) Grinding in mechanical homogeniser
6
Q
Ultracentrifuge
A
- Consists of a rotor housed in a
refrigerated chamber - The rotor is rotated at high speed by an electric motor
- The rotor holds tubes that contain homogenates for fractionation
- The ultracentrifuge is equipped with a vacuum system to reduce friction between the rotor and air. The vacuum prevents heating of the rotor and allows the refrigeration system to maintain the sample at 4C
- Some ultracentrifuges reach speeds over 200,000 rpm, subjecting samples to forces up to 500,000 times greater than gravity (g)
- The centrifugal force causes organelles to move toward the bottom of the centrifuge tube and form a pellet (a process called sedimentation)
7
Q
Polyacrylamide gel electrophoresis
A
- Sodium dodecyl sulfate (SDS) binds to & denatures proteins, giving them the same charge. This means movement across gel depends purely on size
8
Q
Ion exchange chromatography
A
- Separates proteins on charge. Overall charge of protein = ∑ of charges for all its amino acids. When these charges are =, it’s called the isoelectric point. At pH, isoelectric point, proteins have net negative charge, at pH < isoelectric point, net positive charge
9
Q
Size exclusion (gel filtration) chromatography
A
- Separates proteins on molecular size. Can distinguish between monomeric & higher-ordered structures. If protein is part of a large complex, it will fractionate at a greater size
- Proteins in mixture pass through porous beads. Smaller proteins can access the pores more readily than larger proteins. Large proteins pass through entire column length faster & enlarge first, smaller proteins last
10
Q
Affinity Chromatography
A
- Separates proteins based on a specific sequence within the protein. The column has a ligand bound to it that interacts with the sequence
- Proteins in the mixture pass through the column. Must flow right through. But, the specific protein is attracted to ligand & is retained. Specific protein is then eluted by adding an excess of soluble ligand / some other molecule to break interaction - near total purification in 1 step
11
Q
Methods of studying protein interactions
A
- Immunoprecipitation
- Yeast 2-hybrid-reconstitution of a transcription factor
- FRET (Fluorescence Resonance Energy Transfer)
12
Q
Immunoprecipitation
A
- Variation of affinity chromatography using antibodies. Small amonuts of beads with antibodies are incubated w/ cellular extract under non-denaturing conditions. Antibody binds to its antigen & this immune complex is collected by centrifugation. The only protein expected to pellet w/ beads is protein X. If other proteins are detected, indicates that they interact w/ protein X. Western analysis / mass spectrometry can be used to detect co-immunoprecipitating proteins
13
Q
Yeast 2-hybrid-reconstitution of a transcription factor
A
- By the interacting proteins leads to expression of a reporter gene whose activity can be easily measured
14
Q
FRET (Fluorescence resonance energy transfer)
A
- Can be used to examine protein-protein interactions in a cell
- Protein Z fused to CFP & protein Y fused to YFP. If Y & Z interact, then CFP & YFP will interact & FRET will transfer & YFP will fluoresce. Otherwise CFP will fluoresce at its lower wavelength. DNA->mRNA->protein->degraded protein
15
Q
Total magnification
A
- Product of individual lenses
- BUT, more magnification ≠ better, b/c resolving power (resolution is more important)
- Resolution is calculated by how close together 2 objects can be where they can still be seen as separate objects
16
Q
Calculating resolution
A
D=(0.61λ/nsina), where a represents half the angle of the cone of light
λ=wavelength
n= retractive index of the medium between the specimen & the objective
17
Q
3 common types of light microscopy
A
1.) Brightfield microscopy: No contrast other than natural light provided, bad res
2.) Phase contrast: Needs a phase plate to have light of different retractions to increase to increase res
3.) Differential interference contrast (DIC) or Nomarski interference
