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D.Kurry Molecular KCUMB > Analzying cell, molecules and systems > Flashcards

Analzying cell, molecules and systems Flashcards

1
Q

What is a cell culture

A

removal of cells from an organism and promoting their net growth from an favorable artifical environment

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2
Q

WHat is the primary cell culture?

A

Derived directly from animal

Involves enzymatic and or mechanical disruption of tissue and selection steps to isolate the cells of interest from a population

Surivive for a finite peroid of time ; they dont divide so you have to use them

Ex: primary neurons, cardiomyocytes

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3
Q

What are established or continued cell line

A

In which primary culture that has been made immortal by transformation

Most commonly tumor derived or transformed with virus

ex: SH-SY5y (human neuroblastoma dervived)

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4
Q

What the steps for culturing cells?

A
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5
Q

what are the advantages of using cell cultures?

A
  • studying unvaring cell behavior
  • maintained cell charcteristics leading to good reproducibility b/w experiments
  • Controls of growth environment leads to uniformity of sample
  • cultures can be exposed to reagents that stimulate diease or to study pharmacological effects
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6
Q

what are the disadvantages of cell cultures?

A
  • Need to develope / standardize techniques for healthy cell maintainance
  • Quantity is limited
  • dedifferentation and selection can affect the cell biology and biochemistry
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7
Q

What are applications of cell culturing?

A

Research on cell/gene/protein function

Stimulation of a diease in vitro

testing of drugs, vaccine , chemical toxity

chromosomal or genetic analysis

production of biological products (biotech)

regenerative medicine

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8
Q

What is an example of using cell cultures?

A

Cell model of parkinsons disease

  • Expose SH-Sy5Y cells to 6 hydroxydopamine (6-OHDA)
    • Reduce activtity of PMCA (linear loss of activity)
    • Creates reactive Oxygen species
    • triggers apoptosis
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9
Q

What is protein purfication?

A

isolating a single protein from thousands presentin cell

Purfication refers to the study of a proteins structure and function

Two ways to accomplish

  1. Recombinant DNA tech to over express then purify
  2. purfify endogenous proteins
    1. requires sub cellular fractionation - reduces complexity of material
    2. purification
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10
Q

What are differnet types of sub cellular fractionation?

A

Tissue : mechanical blending

homogenate: suspension of different cell types

centrifugation: to separate different cell types based on size and density

Lysis of cells: osmotic shock, ultrasonic vibrations, mechanical blending, forcing through small orifice

Ultracentrifugation : seperate organelles

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11
Q

what are two different types of ultracentrifuge techniques?

A

Fixed angle rotor

Swinging bucket rotor

(test tubes are hanging)

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12
Q

How does speed of centriguation affect a cell homogenate?

A

Lowspeed = pellot that contains ….

  • whole cells
  • nuclei
  • cytoskeletons

medium speed = pellot that contains ….

  • mitochondria
  • lysosomes
  • peroxisomes

Highspeed = pellot that contains ….

  • micrsomes
  • small vesicles

Very high speed =pellot that contains ….

  • ribosomes
  • viruses
  • large
  • macromolecules
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13
Q

What is density gradient centrifugation?

A

density gradient is creatued using sucose or ficoil

the sample is placed on top

it is placed in swinging bucket rotor

The componets will seperate out based on their density (lower densities are higher up in liquid)

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14
Q

How are plasma membranes isolated ?

A

1st centrifudge was 3000rpm x 5min

2nd was 10,000 rmo x 10min

3rd 90,000rpm x 60 min

density - not listed

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15
Q

What happens to PMCA activity and proteins levels as age increases?

A

Although PMCA activity and protein levels declne with increasing age. PCM activity decreases at a faster linear rate

PMCA Activity is Inhibited by Reactive Oxygen Species (ROS)

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16
Q

What inhibits PMCA activity?

A

Reactive Oxygen Species (ROS )

17
Q

What is the different types of column chromatography?

A

based on material in column.

  1. Ion exchange based on charges of stationary phase
    1. if stationary phase is negatively charged bead it will bind to a postively charge species
  2. Gel filtration
    1. Stationary phase filters based on size of the molecule (larger ones come out first)
  3. affinity
    1. bead is specfic to the component your are trying to filter for.
    2. Dr zaidi use this (Calmodulin “ca2+ sensitive” to seperate of Ca pumps on Plamsamembrane )
18
Q

Whata ar ethe techniques used to analyze Proteins?

A

SDS-PAGE

Western Blotting

ELISA

Mass Spect.

19
Q

What is componets used in SDS Page and what is SDS page?

A

Sodium Dodecyl Sulfate - Polacrylamide Gel Eletrophoresis

  • Used for unknown proteins
  • Visual seperated proteins based on stains/dyes
  • ex: commassie blue

SDS

  • largely hydrophobic with a negative charge
  • unfolds proteins gives proteins uniform negative charge
  • allows for all proteins to migrate toward a postive charge in presence of current

beta mercaptoethanol

  • reduces disulfides (furthers denature protein)
20
Q

How is western blotting done?

A
  1. resolve protein samples on native PAGE
  2. fractionated proteins from gel to PVDF membrane (eletrophoretically)
  3. block membrane with neutral protein ( BSA or milk Casein)
  4. Antibodies and (secondary antibodies w/ fluoresence are made )
  5. incubations of membrane with HRP labeled antibody specific to prey protein
  6. Incubate the blot with chemiluminescent HRP subrate and exposed to film

used to identify specific amino-acid sequences in proteins

21
Q

Why are primary/secondary antibodies important to westernblotting?

A

primary antibodies are recongized by secondary antibodies whose already attached to a marker molecule ( fluorescence dyes)

Primary antibody binds to antigen on substrate protein then secondary antibodies w/ mark binds to primary

22
Q

What are enzyme linked immunosorbent Assays?

A
  • ELISA
  • Immunological technique which tests for the levels of specific antigen or antibody concentrations
  • patient serum is probed and test for antiboy and antigen is done conjugated with reactive color enzyme. Color intensity marks presence of enzyme
  • more powerful than western blot is able to quantify its numbers
23
Q

What are the difference b/w indirect and sandwhich (“direct”) methods of ELISA

A

Indirect* = *amount of antibody in sample. where antibody is bound to an antigen from the test

sandwhich = the amount of antigen in sample. where antigen is sandwhiched b/w two antibodies from the test

Ex: pregrancy test which tests for antigen hCG using capture and free antibodys it changes and enforces a color change

24
Q

What is the mechanism for a pregrancy test?

A
  • reaction site
    • where free hCG antibodies binds to hCG in urine and the complex moves to Test site
  • Test Site
    • Immobilized hCG antibody hCG/ab complex from reaction site binds to immobilized hCG antibody. Sandwhich complete dye gives color
  • Control
    • Control site a non specific antibody is immobilized. Dye gives color regardless of +/- of HCG confirms that test is working.
25
Q

What is mass spectromerty?

A

Identifies unknown proteins

Requires tryptic digestion products (peptides fragments), Ionization (charge), a detection method, and computer databases with known protein sizes

26
Q
A

D.Kurry Molecular KCUMB (33 decks)

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