Zaidi Analyzing Cells/Systems 1

Question 1

Define Cell Culture

Answer 1

Refers to the removal of cells from an organism, and promoting their subsequent growth in a favorable artificial environment.

Question 2

Differentiate primary cell cultures and established or continuous cell lines and give examples of each.

Answer 2

PCC: Derived directly from the animal via enzymatic and mechanical disruption of heterogeneous tissue. Survive for a finite period of time. Eg- primary neurons and cardiomyocytes
ECL: a primary culture that has been made immortal by transformation. Eg- SH-SY-5Y human neuroblastoma and Hela cells.

Question 3

Describe the process of Cell Culturing

Answer 3

1 Isolate cells from tissue
2 Grow in culture (aseptic techniques)
3 Sub-culture (passaging) - plate on appropriate surface/dish
4 Use as needed
5 Cryopreservation

Question 4

Advantages of Cell Culture:

Answer 4

Uniform sample
Can expose cells to chemicals/drugs to study pharmacological effects.
Good reproducibility of experiments.

Question 5

Disadvantages of Cell Culture:

Answer 5

Techniques need standardization.
Quantity of material is limited.
Altered cellular pathways due to dedifferentiation and selection.

Question 6

List some applications of cell culture:

Answer 6

Can conduct basic research on the functions of cells/genes/proteins.
Test drugs/vaccines/toxicity.
Disease simulation.
Genetic analysis.
Large scale production of biological products.
Regenerative medicine (stem cells)

Question 7

List an example of a cell culture experiment using 6-OHDA in a model of Parkinson’s disease.

What protein pumps Ca++ out of the neuron after Ca++ influx (influx needed to fuse vesicle to presynaptic membrane)?

Answer 7

6-OHDA creates reactive oxygen species and triggers apoptosis. When exposed to 6-OHDA, PMCA activity decreased in a dose-dependent fashion.

PMCA

Question 8

Why is protein purification important?

List two ways to purify proteins

Answer 8

• It is necessary to study structure and function of individual proteins.
• recombinant DNA technology (over express protein, then purify) and purification of endogenous proteins. Sub-cellular fractionation is usually necessary to reduce the complexity of material before purification.

Question 9

Describe sub-cellular fractionation

Answer 9

Using variable centrifugation speeds to separate different cell types and organelles.

Question 10

Describe density gradient centrifugation

Answer 10

Create a gradient in the solution, suspend the sample, and centrifuge. Then the components are separated by buoyancy or density.

Question 11

What does a synaptosome contain?

Answer 11

presynaptic terminal, postsynaptic terminal, and synaptic gap

Question 12

As age increases, does the activity of PMCA increase or decrease?

Answer 12

Decrease

Question 13

What is column chromatography used for?

List the three common types of column chromatography.

Describe Dr Zaidi’s Research using chromatography:

Answer 13

fractionation of molecules

A- Ion-exchange chrom. (beads are charged and like-charged molecules elute first)
B- Gel-Filtration chrom. (porous beads, small molecules travel in the pores = longer distance traveled = elutes last)
C- Affinity chrom. (substrate of interest attached to bead, binding protein of interest)

Zaidi used affinity chromatography to bind a certain protein to calmodulin, which was covalently attached to the bead. To remove calmodulin from the bead, she used EDTA, a Ca++ chelator, which removed the protein of interest from the calmodulin.

Question 14

List 4 techniques used to analyze proteins:

Answer 14

SDS-Page
Western Blotting
ELISA
Mass Spectrometry

Question 15

What is the purpose of SDS-page?

What is the use of beta-mercaptoethanol?

Answer 15

used to analyze unknown proteins and separates proteins by size
SDS has a negative charge allowing the proteins to migrate towards the positive charge in the presence of current

reduces disulfide bonds, further denaturing the protein

Question 16

How is Western Blotting (immunoblotting) different than SDS-page?

What is the purpose of blocking the membrane with a neutral protein such as BSA or milk casein?

Answer 16

Western Blotting is more focused on specific proteins.

Blocking prevents nonspecific binding of the antibody

Question 17

What does the primary and secondary antibodies do in Western Blotting?

Answer 17

The primary antibody binds to the antigen of interest. The secondary antibody, which is coupled with a fluorescent tag, binds to the primary antibody.

Question 18

Reactive Oxygen Species cause PMCA Aggregation. How was this concluded?

Answer 18

In the presence of beta-mercaptoethanol, the disulfide bonds are reduced. With no beta-mercaptoethanol present, the disulfide bonds are oxidized by AAPH and ACVA.

Question 19

What is the purpose of ELISA and describe the two different kinds. What is the well initially coated with in both kinds?

What is a common application for ELISA?

Answer 19

ELISA quantifies antibody/antigen change.

Indirect ELISA = quantifies antibody change. Well is coated with antigen.
Sandwich ELISA = quantifies antigen change, therefore well is coated with antibody.

Home pregnancy test.

Question 20

Describe the mechanism of home pregnancy tests and the three sites?

Answer 20

1. Reaction site: free hCG antibodies, binds to hCG in urine, moves to test site.
2. Test site: has hCG antibody, hCG/ab complex binds to immobilized hCG antibody, completing the sandwich.
3. Control site: contains non-specific antibody. dye gives color regardless of hCG presence. confirms that the test is working.

Question 21

Describe Mass Spectrometry.

Answer 21

MS identifies protein by utilizing tryptic digestion. This makes peptide fragments. The mass-to-charge ratio of the peptides gives a unique signature that identifies the protein.