Analyzing Cell I

Question 1

Define cell culture

Answer 1

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

Question 2

Define primary cell culture

Answer 2

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

Question 3

What are examples of primary cell cultures?

Answer 3

primary neurons, cardiomyocytes

Question 4

Define established or continuous cell lines

Answer 4

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

Question 5

What are examples of established or continuous cell lines?

Answer 5

SH-SY-5Y; human neuroblastoma derived

Question 6

Describe the basic overview of a cell culture

Answer 6

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

Question 7

What are the advantages of cell culture?

Answer 7

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

Question 8

What are the disadvantages of a cell culture?

Answer 8

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

Question 9

What are the applications of a cell culture?

Answer 9

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

Question 10

Describe the cell model of parkinson’s disease

Answer 10

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

Question 11

Describe protein purification

Answer 11

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

Question 12

Define sub-cellular fractionation

Answer 12

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

reduces the complexity of the material

Question 13

Describe the sub-cellular fractionation

Answer 13

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

Question 14

What is the purpose of ultracentrifugation?

Answer 14

to separate the organelles

Question 15

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

Answer 15

fixed angle

swinging bucket

Question 16

What can be derived following low speed centrifugation?

Answer 16

whole cells
nuclei
cytoskeleton

Question 17

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

Answer 17

mitochondria
lysosomes
peroxisomes

Question 18

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

Answer 18

microsomes

small vesicles

Question 19

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

Answer 19

ribosome
viruses
large macromolecules

Question 20

Describe density gradient centrifugation.

Answer 20

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

Question 21

Describe the isolation of the synaptic plasma membranes

Answer 21

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

Question 22

What is PCMA

Answer 22

ca2+ and ATPase

Question 23

What does reactive oxygen do to the PCMA?

Answer 23

It inhibits the activity

Question 24

Describe column chromatography

Answer 24

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

Question 25

What are the 3 types of column chromatography?

Answer 25

1. ion-exchange 2. gel-filtration 3. affinity

Question 26

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

Answer 26

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

Question 27

Describe ion exchange chromatography

Answer 27

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

Question 28

Describe gel filtration chromatography

Answer 28

separation based on size; pores in the gel

Question 29

Describe affinity chromatography

Answer 29

method of separating based on the interaction of the molecules

Question 30

Describe SDS page

Answer 30

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

Question 31

Describe Western blotting

Answer 31

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

Question 32

What is the common marker used during western blotting?

Answer 32

fluorescent dye

Question 33

Describe ELISA

Answer 33

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

Question 34

What does indirect ELISA measure?

Answer 34

amount of antibody in a sample

Question 35

What does sandwich ELISA measure?

Answer 35

The amount of an antigen in a sample

Question 36

Describe the sandwich ELISA process of pregnancy testing

Answer 36

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

Question 37

Describe the pregnancy test and its 3 different sites 1. reaction sites 2. test sites 3. control sites

Answer 37

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

Question 38

What is mass spectrometry used for?

Answer 38

To identify unknown proteins

Question 39

what does mass spectrometry require?

Answer 39

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