Molecular Methods Dr. Ford

Question 1

Purpose of auxotophy

Answer 1

Nutrient MUST be included in medium.

Question 2

4 principles for cell culture

Answer 2

Food, shelter, climate control, and companionship

Question 3

What does having an antibiotic do for cell culture?

Answer 3

Drug resistance; only cells you will want will grow in presence of antibiotic. Creates SELECTION

Question 4

What is a feeder cell layer?

Answer 4

Layer of fibroblast

Question 5

What are factors of climate control?

Answer 5

Humidity, pH (CO2 is a regulator; acts as a buffer), oxygen (anaerobic vs aerobic, and light

Question 6

What is a symbiotic relationship?

Answer 6

Some cells need other cells to survive; one type of a cell may make nutrients the other cell needs

Question 7

What is contact inhibition?

Answer 7

Change in characteristics due to touching

Question 8

What is quorum sensing?

Answer 8

Cells figuring out if a neighboring cell is friend or foe

Question 9

Some cells can dilute into a single cell and will form colony ie

Answer 9

Colony formation

Question 10

What are the cells for cell mediated immunity?

Answer 10

T cells

Question 11

Humoral immunity is what type of cells?

Answer 11

B cells; makes antibodies but not as much as plasma cells

Question 12

Plasma cells useful for:

Answer 12

Differentiated B cells with pronounced ER to make lots of antibodies

Question 13

T cells, B cells, and plasma cells all work to:

Answer 13

Identify other types of cells

Question 14

Characteristics of an antibody:

Answer 14

“Y” shape= 4 proteins; 2 heavy long chains and 2 light shorter chains. Held together with 4 DISULFIDE bonds

Question 15

Disulfide bonds between cysteines require what type of environment?

Answer 15

OXIDIZING; ie to create antibodies

Question 16

What are some examples of oxidizing environments?

Answer 16

Periplasmic space
OM in prokaryotes
ER lumen
Peroxisomes in eukaryotes

Question 17

What does a reducing environment do to an antibody?

Answer 17

Creates a non-stable antibody ie destroys

Question 18

What is a conserved property for an antibody?

Answer 18

Conserved immunoglobulin domain and conserved Beta strands

Question 19

What is variable in an antibody?

Answer 19

3 binding loops. These loops bind antigens and different from antibody to antibody

Question 20

What kind of bonds for antibody/antigen binding and what properties as well?

Answer 20

Non-covalent (attractive but not permanent) and size and shape are complimentary for binding

Question 21

What are epitopes?

Answer 21

The “fingers” that interact with the antibody. Need to be aware of what epitope and the state of the protein ie folded vs non-folded

Question 22

Mono = 1 epitope only. Expensive of non-expensive?

Answer 22

Expensive

Question 23

Poly = many different antibodies that bind to different epitopes. Expensive or cheap?

Answer 23

Cheap

Question 24

What are MAbs and what are they used for?

Answer 24

Monoclonal antibodies. Herceptin is a MAb shown to bind to HER2 receptors to prevent growth. Targets different aspects of the immune system and prevent inflammation

Question 25

What is the ‘IP’ in ChIP

Answer 25

``` Immuno = antibodies P = precipitation = heavy agarose beads (sink) or magnetic for stickiness; will remove out the of system ```

Question 26

What is ‘Ch’ in ChIP

Answer 26

Chromosome

Question 27

How does ChIP function?

Answer 27

Takes cells and make a covalent interaction with DNA and proteins. Antibody is specific for protein of interest and follows and captures all DNA cross-linked to the antigenic protein.

Question 28

What does formaldehyde do in ChIP?

Answer 28

Formaldehyde connects DNA and protein at non-covalent interactions and also releases covalent binds to see what was captured (DNA)

Question 29

Some examples ChIP is used for include:

Answer 29

Methylation/actelylation of histones and where located in genome. Where polymerase or helicase loads for origin of replication. And transcription factors

Question 30

Rh neg is recessive or dominant?

Answer 30

Recessive

Question 31

Antigen D is:

Answer 31

Rh factor

Question 32

What is agglutination?

Answer 32

Aggregate of antibody and antigens present on on cells = sticks to more than one and creates clumps of cells and antibodies

Question 33

What does anti-D prevent?

Answer 33

Agglutination. Given to Rh mothers to prevent the production of maternal antibodies against fetal Rho/D antigen

Question 34

The MAb: adalimumab/Humira/Amjevita targets what to prevent?

Answer 34

Targets TNF-alpha to prevent inflammation cascade

Question 35

The MAb: Ixekizumab/Taltz targest what to prevent?

Answer 35

Targets IL-171A prevents inflammation by inhibiting binding. Humanized IgG4

Question 36

The MAb: Pembroizumab/Keytruda targets what to release?

Answer 36

Targets PD-1 to release cell cycle repression of T-cells

Question 37

The MAb: Ustekinumab/Stelara targets what to prevent?

Answer 37

Targets IL-17A to prevent inflammation. Human IgG1