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Biochemistry 3380 > Lecture 8 > Flashcards

Lecture 8 Flashcards

1
Q

What is the most sensitive method of protein detection in an SDS-PAGE?

A

radioactivity

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2
Q

What is the sensitivity of radioactivity for protein detection in an SDS-PAGE?

A

most sensitive

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3
Q

What is the specificity of radioactivity for protein detection in an SDS-PAGE?

A

no specificity - meaning it binds all proteins

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4
Q

What are the two types of staining for protein detection in an SDS-PAGE?

A

dye staining; metal staining

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5
Q

What are the three types of dye staining?

A

Ponceau S; Coomassie blue; SYPRO orange

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6
Q

What is the sensitivity of Ponceau S?

A

50ng / mm^2

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7
Q

What is the specificity of Ponceau S?

A

no specificity - meaning it binds all proteins

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8
Q

What is the sensitivity of Coomassie Blue?

A

11.5 ng / mm^2

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9
Q

What is the specificity of Coomassie Blue?

A

no specificity - meaning it binds all proteins

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10
Q

What is the sensitivity of SYPRO Orange?

A

1.5 ng / mm^2

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11
Q

What is the specificity of SYPRO Orange?

A

no specificity - meaning it binds all proteins

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12
Q

What are the three types of metal staining?

A

copper; zinc; silver

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13
Q

What is the most sensitive type of metal staining?

A

silver

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14
Q

What is the sensitivity of copper staining?

A

2.5 ng / mm^2

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15
Q

What is the specificity of copper staining?

A

no specificity - meaning it binds all proteins

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16
Q

What is the sensitivity of zinc staining?

A

2.5 ng / mm^2

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17
Q

What is the specificity of zinc staining?

A

no specificity - meaning it binds all proteins

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18
Q

What is the sensitivity of silver staining?

A

0.4 ng / mm^2

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19
Q

What is the specificity of silver staining?

A

no specificity - meaning it binds all proteins

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20
Q

Generally, what are the two types of immunological staining?

A

polyclonal anti-serum; monoclonal Ab

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21
Q

What is the sensitivity/specificity of polyclonal anti-serum?

A

variable/variable

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22
Q

What is the sensitivity/specificity of monoclonal Ab?

A

variable/very specific

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23
Q

What is the most specific staining method for protein detection?

A

monoclonal Ab

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24
Q

What is the general procedure for Western blotting?

A

reversibly stain membrane with Ponceau S to confirm transfer; blocking membrane in 5% dry milk in TBS; primary antibody probing; secondary antibody probing; final development

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25
Q

Describe the primary antibody probing step.

A

probe with primary antibody/TBS solution; rinse 2-3 times with TBS/Tween 20

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26
Q

Describe the secondary antibody probing step.

A

probe with secondary antibody/TBS solution; rinse 2-3 times with TBS/Tween 20

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27
Q

What are the three standard blotting membranes?

A

nitrocellulose; polyvinylidene difluoride (PVDF); nylon

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28
Q

Describe the use of nitrocellulose.

A

used for general Western blotting; low tensile strength (breaks easily); low protein binding capacity

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29
Q

Describe the use of polyvinylidene difluoride.

A

used for general Western blotting; high chemical resistance/tensile strength; high protein binding and retentive capacity; can be handled

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30
Q

What does it mean for a membrane to be able to be “handled?”

A

can be stripped and reported repeatedly, which is ideal for protein sequencing

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31
Q

Describe the use of nylon.

A

strong synthetic polyamide sheet; can be handled

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32
Q

Which membranes are used for general Western blotting?

A

nitrocellulose; PVDF

33
Q

Which membrane has low tensile strength and low protein binding capacity?

A

nitrocellulose

34
Q

Which membrane has high tensile strength and high protein binding capacity?

A

PVDF

35
Q

Which membranes can be “handled?”

A

PVDF; nylon

36
Q

Ponceau S stains

A

ALL proteins reversibly

37
Q

What does Ponceau S accomplish?

A

determines efficiency of transfer; determines orientation (front/back/up/down); marks MW ladder

38
Q

How do we destain Ponceau S?

A

rinse with ddH2O

39
Q

What are the three standard blocking reagents?

A

5% non-fat dry milk solution; 1% BSA; 3% gelatin

40
Q

Secondary antibodies are conjugated with

A

horseradish peroxidase

41
Q

If a primary antibody binds to the wrong protein, it is called

A

cross-reactivity

42
Q

What is another name for antibodies?

A

immunoglobulins

43
Q

What is an antibody?

A

protein generated by immune system in response to foreign substance

44
Q

What is an antigen?

A

foreign particle that elicits an immune response

45
Q

Draw out the basic structure of an antibody.

A

draw slide 19

46
Q

What is the most common antibody produced by mammals?

A

IgG

47
Q

What are the five antibodies produced by mammals?

A

IgG, IgM, IgA, IgE, IgD

48
Q

Describe the constant regions of antibodies.

A

are the same in all members of a species; differ between species

49
Q

Describe the variable regions of antibodies.

A

are based upon the antigen the antibody recognizes

50
Q

Where specifically on the antibody does the antigen bind?

A

epitope binding site

51
Q

What is an epitope?

A

sequence of 8 to 10 amino acids on the antigen physically recognized by the antibody

52
Q

Who won the Nobel Prize in Physiology/Medicine in 1972?

A

Gerald Edelman and Rodney Porter for discovering the structure of antibodies

53
Q

Who won the Nobel Prize in Physiology/Medicine in 1987?

A

Susumu Tonegawa for discovering antibody diversity

54
Q

What is serum?

A

everything in blood except cells (antibody of interest is absent)

55
Q

What is anti-serum?

A

antibody of interest is present

56
Q

(T/F) There cannot be multiple epitopes on an antigen.

A

FALSE. There can be multiple epitopes on an antigen.

57
Q

B cell + myeloma =

A

hybridoma

58
Q

Describe the longevity of myeloma cells.

A

immortal, because they have an unlimited # of cell divisions

59
Q

Describe the longevity of B cells.

A

mortal, because they have a limited # of cell divisions

60
Q

Each hybridoma will

A

secrete a single IgG

61
Q

Who won the Nobel Prize in Physiology/Medicine in 1984?

A

Niels Jerne; Georges Kohler; Cesar Milstein for theories concerning the specificity/control of immune systems and discovering monoclonal antibodies.

62
Q

Describe the nature of the secondary antibody.

A

polyclonal antiserum

63
Q

Why is the second antibody used?

A

to increase the signal

64
Q

What are the two conjugating detection methods?

A

horse radish peroxidase AND alkaline phosphatase

65
Q

Conjugating detection methods can detect what two types of reactions?

A

chemiluminescent and colorimteric

66
Q

In chemiluminescent reactions, HRP detects

A

luminol

67
Q

In chemiluminescent reactions, AP detects

A

CDP-star

68
Q

In colorimetric reactions, HRP detects

A

4CN - purple; DAB - brown; TMB - purple

69
Q

In colorimetric reactions, AP detects

A

BCIP/NBT - purple

70
Q

What is the main difference between staining and immunological detection?

A

staining isn’t specific, immunological detection is specific because antibodies will bind to the epitope on the antigen

71
Q

What is the purpose of incubating the membrane with BSA, gelatin, or a mixture of dry milk?

A

used to block areas of nitrocellulose that are not already bound by target protein

72
Q

What is the difference between a primary antibody and a secondary antibody?

A

Primary antibody binds specifically to target protein; Secondary antibody binds specifically to primary antibody

73
Q

What is the purpose of the Tween 20 used in the wash steps?

A

Non-ionic detergent; Interrupts any non-specific protein-protein binding

74
Q

What is the advantage of using colorimetric enzyme (like horseradish peroxidase) conjugated to a secondary antibody as opposed to being conjugated to the primary antibody?

A

With only primary antibody, colorimetric detection would be impossible and we would see nothing – 2 antibody systems usually use a polyclonal secondary antibody which allows for a lot of binding and therefore will amplify signal of detection

75
Q

What are the advantages and disadvantages of using a chemiluminescent reaction versus a colored precipitate reaction?

A

Chemiluminescent is expensive, but extremely sensitive and can be exposed as many times as needed; Colored precipitation is inexpensive , but less sensitive and once overexposed is ruined

76
Q

What is the difference between monoclonal and polyclonal antibodies?

A

Polyclonal antibodies are mixtures of antibodies where each recognizes different epitopes of antigen. Whereas monoclonal antibodies recognize only 1 epitope.

77
Q

Define pre-immune serum.

A

serum before introduction of Ag

78
Q

Define cross reactivity.

A

some Ab will recognize Ab on other Ag because of similar epitope.

Biochemistry 3380 (9 decks)

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