Lecture #2 - Cell Culture & Protein Experiments

Question 1

How can cells be isolated from intact tissues?

Answer 1

Disrupt ECM and cell-cell junctions in two ways:

1. With proteolytic enzymes (trypsin/collagenase) to digest proteins
2. With EDTA to chelate Ca2+ on which cell-cell adhesions depend

Question 2

FACs

Answer 2

Fluorescent activated cell sorter
Cell have receptors on surface that Ab attach to
Separate cell types from mixed suspension

steps:
1. Labeled cells can be separated from unlabeled cells via Ab specific to the cell
2. Machine puts a + or - charge on ladled cells and separate based on charge

Question 3

In vitro

Answer 3

In culture

Question 4

In vivo

Answer 4

In the organism

Question 5

Primary cell cultures

Answer 5

Cells grown in a disk or flask

Prepared directly from tissues

Question 6

What is the main issue with using primary cell cultures?

Answer 6

The cells eventually die off due to the shortening of telomeres, called replicative cell senescence

Question 7

Immortilized cell line

Answer 7

Used by many labs
Cells become immortalized due to oncogenes
Do not die off as long as they have nutrients

Question 8

Contact inhibition

Answer 8

Cells that collide stop dividing

Question 9

Oncogenes

Answer 9

Mutated protooncogene

Misses checkpoints

Question 10

Protooncogenes

Answer 10

Normal cell activity

Question 11

Cancer cells do not have _______, and grown on top of one another.

Answer 11

contact inhibition

Question 12

Transformed cell lines are generated from _______ cells and can be _______.

Answer 12

cancer; frozen

Question 13

What is the main problem with using immortalized cell lines?

Answer 13

They don’t function the same way as normal cells, so it is difficult to get the same results with normal cells
Can’t publish research using these cells

Question 14

How can cells be separated into their components?

Answer 14

1. Cells broken up by osmotic shock or ultrasound vibration

2. Organelles are separated by size and density using the prep. ultracentrifuge

Question 15

Low speed ultracentrifugation

Answer 15

Large components sediment and form a pellet

Nuclei, whole cells, cytoskeletons

Question 16

Medium speed ultracentrifugation

Answer 16

Medium components sediment and form a pellet

Mitochondria, lysosomes, peroxisomes

Question 17

High speed ultracentrifugation

Answer 17

Small components sediment and form a pellet

Robosomes, viruses, large macromolecules (protein, DNA, RNA)

Question 18

During ultracentrifugation, the temperature must be kept _______ to avoid _______.

Answer 18

low; denaturing

Question 19

Cells can be used as _______ to produce certain _______.

Answer 19

factories; proteins

Question 20

How do we manipulate cells to produce large amounts of certain proteins?

Answer 20

Recombinant DNA technology

Use expression vectors

Question 21

Expression vectors

Answer 21

Special plasmids that allow the cell to make a lot of a specific protein
Can delay the production of protein to avoid toxicity

Question 22

Expression vectors can delay production of protein so that the cell will not be damaged due to _______.

Answer 22

toxicity

Question 23

Proteins can be separated by _______.

Answer 23

column chromatography

Question 24

What are the four types of column chromatography?

Answer 24

1. Ion-charge chromatography
2. Hydrophobic chromatography
3. Gel-filtration chromatography
4. Affinity chromatography

Question 25

Ion-charge chromatography

Answer 25

Based on charge of proteins
Small beads with a + or a - charge
Proteins fractionate according to surface charge
Not commonly used

Question 26

Hydrophobic chromatography

Answer 26

Based on hydrophobicity of proteins

Not commonly used

Question 27

Gel-filtration chromatography

Answer 27

Based on size of proteins
Proteins are separated by size
Column is packed with tiny porous beads
Small proteins enter the pores and linger inside beads as they pass down
Larger proteins travel down quickly, uninhibited

Very common, very useful

Question 28

Affinity chromatography

Answer 28

Based on specific binding sites of proteins
Sustrate molecule covalently coupled to inert matrix
Protein that binds to substrate specifically is retained by the matrix
Can use antibody or ligand
Can also use expression vectors to add a tag and then use this method

Question 29

Using _______, any gene can be modified to produce its _______ with a special recognition tag.

Answer 29

recombinant DNA technology; protein

Question 30

GST tag

Answer 30

Special recognition tag
Bonds to glutathione
Large and may inhibit interactions

Question 31

SDS-PAGE

Answer 31

SDS-PolyAcrylamide Gel Electrophoresis
Separates proteins
Electric field applied causes protein to migrate depending on size and shape

Question 32

What makes the pores in SDS-PAGE? What is its relationship to the size of pores?

Answer 32

Polyacrylamide. The more polyacrylamide, the smaller the pores.

Question 33

SDS

Answer 33

A detergent that unfolds the protein (denatures it) by binding to hydrophobic regions
Makes the protein linear and adds a - charge

Question 34

β-mercaptoethanol

Answer 34

Breaks disulfide bonds (S-S) in proteins

Added in addition to SDS in SDS-PAGE

Question 35

What stain binds to the proteins on a gel electrophoresis?

Answer 35

Coomassie blue

Question 36

The _______ blot technique can be used after SDS-PAGE to detect a specific protein.

Answer 36

Western

Question 37

Western blot

Answer 37

Technique used to detect proteins
Proteins on the gel are transferred to a membrane and then incubated with the antibody for that protein
The interaction is identified using biochemical methods

Question 38

You have the antibody for protein y, how do you identify other proteins that bind to protein Y?

Answer 38

Use affinity chromatography
The antibody will recognize and specifically bind to protein Y
If the associated proteins also bind to protein Y, then it will be bound to protein Y that is bound to the antibody