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Molecular Biology > Exam 2 content > Flashcards

Exam 2 content Flashcards

1
Q

Proteins are polymers of……

A

amino acids

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2
Q

Peptide bonds form through [..]
reactions

A

dehydration

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3
Q

Green fluorescent protein (GFP) is a
bioluminescent protein that…..

A
  • converts blue light to green light
  • is used as a marker protein for many many experiments (it plays the same role in protein studies as a conjugated antibody)
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4
Q

What determines protein shape?

A

many weak and strong chemical bonds

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5
Q

What are Chaperones?

A
  • proteins that assist in folding or refolding of a protein
  • many are referred to as heat shock proteins
  • EX) Hsp70 helps fold ER proteins
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6
Q

What is denaturation?

A

process of unfolding a protein causing it to lose 3D shape permanently

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7
Q

Example of a reducing agent

A

beta-mercaptoethanol, break disulfide bonds, covalent bonds that form between sulfur atoms

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8
Q

Examples of quaternary structure proteins

A

Hemoglobin
Insulin

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9
Q

What is a motif?

A
  • short amino acid sequence with a function
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10
Q

What is a protein domain?

A
  • segment of protein that folds into a stable structure independent of the rest of the protein
  • usually 50-300 consecutive amino acids residues in length

EX) immunoglobin domain folds in antibodies

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11
Q

What are Src proteins?

A
  • proteins that contain a kinase catalytic domain and a regulatory region, SH2 and SH3 domains
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12
Q

What are some ways proteins can get cleaved?

A

-Removal of the first amino acid residue, methionine (all proteins)
-Proteolytic cleavage of a signal sequence (membrane or secreted proteins)
- Proteolytic cleavage

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13
Q

What is the purpose for cell isolation?

A

to allow primary cells to grow in a culture

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14
Q

Primary cells vs. Cell lines

A
  • Primary cells are isolated directly from an
    individual.
  • Cell lines have been “transformed” to grow in
    culture indefinitely.
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15
Q

What is Flowcytometry?

A

the study of cell populations based on their light scattering properties

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16
Q

What components are a part of flow cytometry?

A
  • Forward scatter or FCS: measure scatter along the path of the laser
  • Side scatter or SSC: measures scatter at a ninety-degree angle relative to the laser
  • photodiode converts the light into an electrical signal and the intensity of the produced voltage is proportional to the size(FS) or complexity(FS) of the cell
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17
Q

How does flow cytometry work?

A

(1) cells from single cell suspension with a specific antibody are moved through a thin pipe
(2) light is shot through the thin pipe and measured using a forward and side detector

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18
Q

What is cell homogenization?

A

gaining access to the proteins by breaking cell membrane in a controlled fashion

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19
Q

What are some examples of cell homogenization?

A

Physical: sonification, french press, mortar/pestle, freeze/thaw

Chemical: detergent (solubilize), enzymes (lysozyme)

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20
Q

What is the purpose of cell fractionation?

A
  • to separate centrifuged cell components by density
  • Larger/denser components move to the bottom of the tube while smaller /less dense components remain in suspension (supernatant)
  • Repeated centrifugation at progressively higher speeds will fractionate cellular components
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21
Q

advantage’s of using primary cells…

A
  • used for physiological studies (not transformed)
  • respond to signals appropriately
22
Q

disadvantage’s of using primary cells…

A

can only replicate so many times before they stop responding to signals appropriately

23
Q

advantage’s of cell lines…

A

are “transformed” cells that grow in culture indefinitely

24
Q

disadvantage’s of cell lines…

A

do not respond to signals appropriately

25
Q

Which type of light scatter measures size? (forward or side)

A

forward

26
Q

Which characteristic is interrogated by side scatter?

A

cell complexity

27
Q

Why are protein samples boiled prior to loading an SDS gel?

A

to denature them

28
Q

Why is SDS added to the samples and the gel?

A

to denature the proteins and to give them a uniform net negative charge

29
Q

Why is a reducing agent added to the
samples?

A

to further denature the protein by breaking disulfide bonds

30
Q

Which type of light scatter (forward or side) is
associated with cellular complexity?

A

side scatter

31
Q

What is the difference between a primary and
secondary antibody?

A

A primary antibody binds to a specific antigen, and a secondary antibody binds to the primary antibody

32
Q

What is the difference between a nucleoside
and a nucleotide?

A

nucleoside - contains a 5-carbon sugar and a nitrogenous base ONLY

nucleotide - contains a 5-carbon sugar, nitrogenous base, and 1-3 phosphate groups

33
Q

List the three components that make up the building blocks of nucleic acids?

A

Phosphate group(s), 5-carbon sugar, nitrogenous base

34
Q

How does the 5-carbon sugar differ between DNA and RNA?

A
  • DNA contains a ‘deoxy’ ribose with a H group instead of a OH group on the 2’ carbon
  • RNA is a regular ribose with an OH on the 2’ carbon
35
Q

Which nitrogen-containing base differs between DNA and RNA?

A

DNA uses thymine

RNA uses uracil

36
Q

What is hyperchromicity?

A

When absorbance at a specific wavelength increases as temperature increases

37
Q

How is the Tm determined for a DNA molecule?

A

By finding the midpoint on an absorbance vs temp graph.

The midpoint indicates when half of the DNA is double-stranded and the other half is single-stranded.

A characteristic of Tm is 1) G:C content of a DNA molecule and 2)
the ionic strength of the solution that the DNA molecule is in.

38
Q

Why does a high salt buffer increase the Tm of a
DNA molecule?

A

It stabilizes the DNA which makes it harder to separate the DNA strands

39
Q

Describe the sequence of events that take place to activate a Src family kinase

A

A phosphate group is removed by a phosphatase. It undergoes conformational change releasing its SH2 domain and allowing it to bind to an activating enzyme, via both its SH2 and SH3 domains. It is now a functioning kinase that can phosphorylate other substrates right after it phosphorylates itself

40
Q

explain how DNAse was used to determine the number of nucleotides in one turn of the double helix

A

In the Mica experiment they laid DNA(neg. charged) on a salt surface(pos. charged) and added DNAse. The enzyme could only degrade the regions on DNA not attached to the salt surface. The intact DNA that was left was put through gel electrophoresis and the banding pattern was the result of the the nucleotides at each turn of the DNA

41
Q

explain why more information is gleaned from the major groove (rather than minor groove) of a DNA molecule

A

because there are more observable patterns on the major groove than on the minor groove

42
Q

explain why the OD 260 of single stranded DNA absorb more UV light than double stranded DNA

A

Base stacking diminishes the capacity of the
bases to absorb light, therefore more light being absorbed through single stranded DNA

43
Q

What could you do to determine the melting temperature of a DNA sequence?

A

Take into account the G:C content of the sequence and the ionic strength of the DNA solution

44
Q

What is the purpose of nucleic acid isolation?

A

get starting material for sequencing or template for PCR or cloning

45
Q

What are the 3 options and the 3 steps for nucleic acid isolation?

A

Options: organic extraction, spin baskets, magnet particles
3 steps: cell lysis, contaminant removal, nucleic acid purification

46
Q

What is the purpose of plasmid DNA isolation?

A

To isolate plasmid DNA and not genomic DNA

47
Q

What is gel electrophoresis used for?

A

Separate molecules based on size.

48
Q

What is Ethidium Bromide

A

Aromatic compound used in gel electrophoresis to detect nucleic acids.
Excited by UV light and emits light at 600 nm.
Shows up as bright band on black background.

49
Q

How does gel electrophoresis work?

A

DNA samples are loaded
onto an agarose gel in an
ionic buffer and current is
applied, pushing nucleic
acids through the gel.
Nucleic acids move toward
the positive electrode, with
smaller molecules moving
faster than larger ones.

50
Q

What is quantification?

A

Molecular Biology (3 decks)

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