Lecture 21 Flashcards
Define the characteristics of a primary cell culture. give an example.
derived directly from the animal
ensymatic/mechanical treatment is used to isolate the cell of interest from a heterogenous sample
usuallys “survive for a finite period of time”
ex. primary neurons or cardiomyocytes
Define the characteristics of an established/continuous cell line culture. give an example
a primary culture that has been made immortal via transformation
Usually tumor derived or transformed with a virus
ex. SH-SY-5Y (human neuroblastoma derived), and many other cancers
What are some advantages of culturing cells?
study of cell behavior without complexity/variation that occurs in nature
Good reproducibility between experiments
Uniformity of sample
researchers have total control over exposures (to simulate diseases/study pharmacological effects)
what are some disadvantages of culturing cells?
techniques must be developed/standardized to maintain healthy cells
Quantity of material is limited
Dedifferentiation and selection may occur and impact the original cellular mechanisms/pathways
To create a cell model of Parkinson’s disease, you would expose _____ cells to 6-OHDA in order to create reactive oxygen species that trigger apoptosis/decrease PMCA
SH-SY5Y
what does PMCA stand for?
plasma membrane Ca2+ ATPase (calcium pump that uses ATP to keep the Ca2+ gradient in neurons) that pumps Ca2+ out of the neuron
How is 6-OHDA (6-hydroxydopamine) created?
dopamine is converted to 6-OHDA
_____ is crucial to be able to study the unique structure/function of INDIVIDUAL proteins, and this process may also use _____ DNA technology to over express a protein
Purification
Recombinant
What is commonly done before purification of a protein, in order to reduce the complexity of the material in a sample?
sub-cellular fractionation
When it comes to sub-cellular fractionation, what is homogenate?
suspension of different cell types (usually achieved via mechanical blending of a sample
When it comes to sub-cellular fractionation, give some examples of processes used to achieve lysis of cells
osmotic shock, ultrasonic vibration, mechanical blending, and forcing the sample through a small orifice (not important)
When it comes to sub-cellular fractionation, what is ultracentrifugation used to achieve?
the separation of organelles
describe the sequential process of sub-cellular fractionation
mechanical blending
centrifugation of the homogenate to separate based on size/density
lysis of cells
ultracentrifugation
what is the difference between a fixed angle rotor and a swinging bucket rotor centrifuge.
fixed angle: test tubes stay in the same position
Swinging bucket rotor: test tubes have a hinge at the top and swing parallel to the floor (like a helicopter)
describe what occurs, in terms of what collects at the bottom of the test tube, at low-speed, medium-speed, high-speed, and very-high-speed centrifugation.
LS: whole cells, nuclei, and cytoskeletons
MS: mito, lysosomes, and peroxisomes
HS: microsomes, and small vesicles
VHS: ribosomes, viruses, and large macromolecules
describe density gradient centrifugation and how low-buoyant density and high-buoyant density substances are collected in high concentrations for testing
low-buoyant density components collect in a line towards the top of the test tube, after centrifugation
high-buoyant density components collect in a line towards the bottom of the test tube, after centrifugation
several test tubes worth of the different buoyancy components are collected and concentrated into separate test tubes
what happens to PMCA activity with age? how is PMCA activity inhibited?
it declines
reactive oxygen species (ROS)
give a brief, overall description of column chromatography
a solid matrix is loaded into a tube with a porous plug at the bottom
a sample is applied to the top of the matrix from a large reservoir
the different molecules in the sample are separated by various properties by the matrix
In terms of column chromatography, compare Ion-exchange, gel-filtration, and affinity matrices.
Ion-exchange: depending on the charge of the matrix beads, oppositely charged particles will stick to the bead and the rest of the sample passes through
Gel-filtration: mechanical separation where small-enough molecules become “retarded” in a porous bead
Affinity: Enzyme affinity covalently attaches enzymes to substrates, that are moving through the column, to the bead
what type of beads would be used in affinity chromatography to separate PMCA from a sample?
calmodulin sepharose beads
List the 4 techniques used to analyze proteins
SDS-PAGE
Western Blotting
ELISA
Mass Spectometry
describe the purpose of SDS PAGE
It’s purpose is to separate proteins based on their size (smaller (lighter) moves faster and larger(heavier) moves slower)
describe the mechanism of SDS PAGE
SDS is a hydrophobic and negatively charged treatment that unfolds proteins and gives all proteins in a sample a uniform charge
Beta-mercaptoethanol: reduces the disulfide bonds between the subunits in some proteins (separates subunits)
Polyacrylamide-gel electrophoresis: sample loaded at the cathode (-) end of gel and is electrophoresed towards the Anode (+) end to separate proteins
Why is SDS, when used for SDS-PAGE, used to give all proteins present in a sample a uniform charge?
if this did not occur, the electrophoresis part of SDS-PAGE analyzation would separate proteins based on their size AND their various charge types and the amount of charge
it would be much more confusing than just separating based on size, so SDS makes the electrophoresis more easily interpreted
