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Molecular Exam 2 > Analyzing Cell I > Flashcards

Analyzing Cell I Flashcards

1
Q

Define cell culture

A

refers to the removal of cells from an organism and promoting their growth in a favorable artificial environment

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2
Q

Define primary cell culture

A

derived directly from the animal
enzymatic and or mechanical disruption of the tissue to isolate the tissue
survive for a finite period of time

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3
Q

What are examples of primary cell cultures?

A

primary neurons, cardiomyocytes

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4
Q

Define established or continuous cell lines

A

a primary culture that has been made immortal by transformation
most commonly tumor derived

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5
Q

What are examples of established or continuous cell lines?

A

SH-SY-5Y; human neuroblastoma derived

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6
Q

Describe the basic overview of a cell culture

A
  1. isolate the tissue; revive frozen cell population
  2. grow in culture using aseptic techniques
  3. subculture (passaging); plate on an appropriate surface
  4. count cells
  5. use as needed
  6. cryopreservation
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7
Q

What are the advantages of cell culture?

A
  1. study cells without the complexities of life
  2. can maintain several generations
  3. uniformity of the samples
  4. can expose cultures to reagents
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8
Q

What are the disadvantages of a cell culture?

A
  1. need to develop techniques in order to maintain healthy cells
  2. quantity limited
  3. dedifferentiation and selection
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9
Q

What are the applications of a cell culture?

A
  1. can conduct basic science
  2. simulation of a disease
  3. testing of drugs
  4. chromosomal analysis
  5. biological products
  6. regenerative medicine
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10
Q

Describe the cell model of parkinson’s disease

A
  1. expose SH-SY5Y cells to 6-OHDA
  2. reactive oxygen species
  3. apoptosis
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11
Q

Describe protein purification

A

purification is able to study the function of individual proteins
by…
recombinant DNA technology, endogenous proteins

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12
Q

Define sub-cellular fractionation

A

remove all of the cells just to get the protein of interest

reduces the complexity of the material

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13
Q

Describe the sub-cellular fractionation

A

tissue: mechanical blending
homogenate: suspension of different cell types
centrifuge to separate different cell types
lysis of cells
ultracentrifugation

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14
Q

What is the purpose of ultracentrifugation?

A

to separate the organelles

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15
Q

What are the two different types of rotors that can be used in preparative centrifugation?

A

fixed angle

swinging bucket

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16
Q

What can be derived following low speed centrifugation?

A

whole cells
nuclei
cytoskeleton

17
Q

What can be derived following the supernatant exposure to medium speed centrifugation?

A

mitochondria
lysosomes
peroxisomes

18
Q

What is able to be derived from the supernatant following to the high speed centrifugation?

A

microsomes

small vesicles

19
Q

What is able to be derived after the supernatant is subjected to very high speed centrifugation?

A

ribosome
viruses
large macromolecules

20
Q

Describe density gradient centrifugation.

A

The layers sediment and the low buoyancy is above the high density component

21
Q

Describe the isolation of the synaptic plasma membranes

A
  1. homogenize the tissues derived from he brain
  2. centrifuge the sample at a low speed
  3. save the supernatant
  4. centrifuge that at a medium speed
  5. divided into supernatant and pellet
  6. centrifuge the supernatant at a high speed and get microsomes
  7. or take the pellet and homogenize and apply density gradient centrifugation to get synaptosomes
  8. kyse the synaptosomes
  9. SPMs result
22
Q

What is PCMA

A

ca2+ and ATPase

23
Q

What does reactive oxygen do to the PCMA?

A

It inhibits the activity

24
Q

Describe column chromatography

A

The sample is applied and a solvent is continuously added to the sample and used out of the column at different speeds to receive the fractionated molecules

25
Q

What are the 3 types of column chromatography?

A
  1. ion-exchange
  2. gel-filtration
  3. affinity
26
Q

What are the 4 techniques that can be used to analyze proteins

A
  1. SDS-PAGE
  2. Western blotting
  3. ELISA
  4. Mass spectrometry
27
Q

Describe ion exchange chromatography

A

Using the ionic characteristics of the target molecule to get it to stick to the beads or vice versa

28
Q

Describe gel filtration chromatography

A

separation based on size; pores in the gel

29
Q

Describe affinity chromatography

A

method of separating based on the interaction of the molecules

30
Q

Describe SDS page

A

The sample is loaded into the electrophoresis apparatus
The negatively charged molecules react with the SDS molecules and move down the porous gel complex

based in weight

31
Q

Describe Western blotting

A
  1. The sample is allowed to denature
  2. Gel electrophoresis
  3. Antibodies are applied to the sample and washed
  4. secondary antibodies are applied
  5. stained to see these antibodies to find a target protein
32
Q

What is the common marker used during western blotting?

A

fluorescent dye

33
Q

Describe ELISA

A

tests for the levels of a specific antigen or antibody concentrations using a corresponding antibody

probe the patient serum with test antibody or serum
-antigen is conjugated to receive the enzyme and a color change is observed if the antibody is present

34
Q

What does indirect ELISA measure?

A

amount of antibody in a sample

35
Q

What does sandwich ELISA measure?

A

The amount of an antigen in a sample

36
Q

Describe the sandwich ELISA process of pregnancy testing

A

A free monoclonal antibody for hCG binds to the monoclonal antibody that has captured the hCG and traps it (hCG) in between the two monoclonal antibodies which results in a color change

37
Q

Describe the pregnancy test and its 3 different sites

  1. reaction sites
  2. test sites
  3. control sites
A
  1. reaction site:
    - free hCG antibodies
    - bind to the hCG that is present in urine
    - complex then moves to the test site
  2. test site:
    - has immobilized hCG antibody
    - hCG/ab complex from the reaction site
    - binds to immobilized hCG antibody
    - sandwich is complete and dye gives a color
  3. Control site:
    - nonspecific antibody is immobilized
    - dye gives color regardless of hCG and confirms that the test is working
38
Q

What is mass spectrometry used for?

A

To identify unknown proteins

39
Q

what does mass spectrometry require?

A

tryptic digestion products (peptide fragments), ionization (charge), and a detection method, computer

Molecular Exam 2 (13 decks)

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