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Biol 2020 Lab Flashcards

1
Q

When do Artemia release cysts?

A

During times of harsh environmental stress

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2
Q

State of low metabolism in cysts?

A

Diapause

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3
Q

Cysts are resilient to: (3)

A

Anoxia, desiccation, sub-zero temps

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4
Q

Purpose of HSPs

A

Binding to proteins to prevent denaturation

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5
Q

Cysts survive diapause due to:

A

HSPs

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6
Q

Which HSP is most prevalent?

A

HSP90

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7
Q

What’s the pipetting range of a P20?

A

1-20 ul

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8
Q

What’s the pipetting range of a P200?

A

20-200 ul

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9
Q

What’s the pipetting range of a P1000?

A

200-1000 ul

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10
Q

What colours (2) are the P20/P200 tips?

A

Yellow or Clear

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11
Q

What colour are the P1000 tips?

A

Blue

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12
Q

What sort of medium is required for the Bradford Assay?

A

Acidic

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13
Q

What does the Bradford reagent do?

A

Bind to proteins

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14
Q

What is the colour change when the Bradford reagent is activated?

A

Brown to Blue

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15
Q

What does BSA stand for?

A

Bovine Serum Albumin

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16
Q

What is BSA used for in the Bradford Rxn?

A

Standard to compare the Artemia samples to

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17
Q

What substance are proteins usually electrophoresed through?

A

Polyacrylamide

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18
Q

What is necessary to observe electrophoresed proteins to determine if a band was present in the studied location?

A

Coomassie Blue Stain

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19
Q

What detergent is used in electrophoresis?

A

SDS (Sodium Dodecyl Sulfate)

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20
Q

What is, what is the purpose of (2), and when do we use SDS?

A

It is a detergent
It disrupts the 2nd structure of proteins, denaturing them
It coats proteins with - charges to mask R group charges
Used in electrophoresis

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21
Q

What is DDT (mercaptoethanol)?

A

Reducing agent

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22
Q

What is the acronym for mercaptoethanol and what does it do?

A

DDT

Breaks disulfide bonds

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23
Q

When is DDT used?

A

Before running electrophoresis

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24
Q

How can protein migration be plotted?

A

Log Molecular Weight vs distance travelled

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25
Q

What are the components of the sample buffer?

A

SDS, glycerol, DDT, tracking dye

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26
Q

What gel is used?

A

Bolt Bis-Tris Plus

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27
Q

What is the [polacrylamide] of the gel?

A

12%

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28
Q

What were labelled using immunoflorescence? (2)

A

Microtubules and nuclei

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29
Q

What kind of cells were used for immunoflorescence?

A

HEK (Human embryonic kidney)

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30
Q

What does HEK stand for?

A

Human embryonic kidney

31
Q

What are the secondary antibodies in immunoflorescence tagged with?

A

Flourochromes

32
Q

What flourochrome was used to label microtubules?

33
Q

What flourochrome was used to tag DNA?

34
Q

What does the primary antibody in immunoflorescence recognize?

35
Q

What is the name of the primary antibody used in immunoflorescence?

A

Mouse anti-tubulin

36
Q

What color is FITC?

37
Q

What color is Hoechst’s?

38
Q

What does Hoechst’s bind to?

39
Q

What color is Alexa Fluor 555?

40
Q

What flourochrome is red?

A

Alexa Fluor 555

41
Q

What is the role of methanol? (3)

A

Fixes cells so cytoskeleton stays intact
Helps cells stick to coverslip
Permeabilize the cell membrane

42
Q

What do humidity chambers do?

A

Provides moist environment so cells don’t dry out

43
Q

What is the name of the secondary antibody?

A

Rabbit anti-mouse, conjugated to FITC

44
Q

What does glycerol do?

A

Causes the sample to sink to the bottom of the gel electrophoresis wells

45
Q

What does TBS-Tween do?

A

Protein protecting buffer

46
Q

What protein protecting buffer was used to protect the nitrocellulose membrane proteins?

47
Q

What is the purpose of tracking dye?

A

Visibly electrophorese ahead of the proteins

48
Q

What class of antibodies were used and where do they come from?

A

IgG-Class, animal host

49
Q

How are polyclonal antibodies produced? (2)

A
  1. Antigen injected

2. Antibodies for different epitopes of antigen harvested and used

50
Q

How are monoclonal antibodies produced? (2) What is a benefit over polyclonal?

A
  1. Antigen injected
  2. Antibody producing cells removed and cloned

More specific, less likely to bind to other proteins

51
Q

What is HRP and what is it used in?

A 
Horseradish Peroxidase (Enzyme)
Used in chemiluminescence
52
Q

What is the secondary antibody in chemiluminescence tagged with?

53
Q

What does HRP do and in what conditions?

A

Catalyses the oxidation of luminol in the presence of an oxidising agent => blue light

54
Q

What is Ponceau stained used for?

A

Temporarily mark protein lanes red for labelling pre Western blotting

55
Q

What is the milk solution used for?

A

Coats nitrocellose in casein to minimize non-specific binding of the primary antibody

56
Q

What are the steps of Kohler Illumination

A
  1. Turret to O
  2. Place contrasty slide
  3. Focus w/ 10x objective
  4. Close the field iris
  5. Focus light by moving substage condenser mount
  6. Center the ring of light using screws
  7. Open field iris to fill the field of view
57
Q

At what temperature must mammalian cells be grown?

58
Q

What [CO2] do mammalian cells require?

59
Q

What does cryo media contain to slow the freezing rate of cells?

A

DMSO (demethylsulfoxide)

60
Q

What does DMSO do?

A

Slow the freezing rate of cells

61
Q

What is used to minimize contamination of cell cultures?

A

Penecillin & Streptomysin

62
Q

Why is media pink?

A

Phenol Red indicator

63
Q

What colour does cell culture media turn if it becomes acidic?

64
Q

What colour does cell culture media turn if it becomes basic

65
Q

What is passaging or subculturing?

A

Moving cells to fresh media

66
Q

What is it called when cells are moved to a fresh media

A

Subculturing

67
Q

What is 100% confluence?

A

When cells make a solid monolayer on the substrate

68
Q

Where is trypsin formed and what does it do?

A

Produced in the pancreas, dissociates cells from the substrate by dissolving attachment points

69
Q

What does Trypan blue do?

A

Stains only dead cells blue (bc they’re permeable)

70
Q

What does PBS do?

A

Removes Calcium

71
Q

What is Bright Field “O” good for?

A

Coloured cells (trypan blue stained dead cells)

72
Q

What is Phase Contrast “PhN” used for?

A

Seeing otherwise transparent cells

73
Q

What wavelength is used in the spectrophotometer?

Biol 2020 (2 decks)

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