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BMSc - Labs > Western Blot > Flashcards

Western Blot Flashcards

1
Q

What is Western blotting used for?

A

To detect the presence of a specific protein in a protein mixture extracted from cells
To evaluate the size of a protein
Measure the amount of protein present

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2
Q

What are the three steps in western blotting?

A

Separation by size
Protein transfer to solid support
Detection of target protein

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3
Q

How are antibodies for Western blot made?

A

Antigen injected into animal and its immune system produces a response

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4
Q

What do polyclonal antibodies consist of?

A

Mixed pool of Ig molecules

These bind to different epitopes found on a single antigen

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5
Q

What do monoclonal antibodies bind to?

A

A single epitope on the target antigen

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6
Q

Which antibody type has a higher probability of detecting the protein?

A

Polyclonal

Bc binds to various segments

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7
Q

Which antibody type is more sensitive?

A

Polyclonal

Bc amplification occurs are every binding site

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8
Q

What can cell lysates be prepared from?

A

Transfected cells carrying a protein expression vector
OR
immortalised cell lines known to express the target protein

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9
Q

At which temp should cell lysates be prepared?

A

Cold/on ice

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10
Q

Why may sonication be required?

A

To fully release the protein from certain cell and tissue samples

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11
Q

What is present in the gel of most western blots?

A

SDS

Reducing agent

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12
Q

Why does SDS-PAGE work?

A

The proteins will be negatively charged due to the SDS
They then migrate in an electric field through the gel to the positive electrode
The mass:charge ratio gets normalised by the binding to SDS along the gel
Bigger proteins then go further

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13
Q

How are the proteins transferred onto a blotting membrane?

A

An electric field is run through perpendicular to the angle of the gel

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14
Q

What can be added to the gel running buffer to help the proteins bind to the blot?

A

Methanol

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15
Q

What are the advantages of using a nitrocellulose gel?

A

Excellent protein binding and retention capabilities

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16
Q

What are the disadvantages of nitrocellulose gel?

A

Brittle and less effective if re-use needed

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17
Q

Why are gloves always worn in Western blot?

A

Oils in hands can interfere with the signal on the blotting membrane

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18
Q

Why only one band visible in Western blot film?

A

Antibodies should only bind to that protein

The unbound is then washed off

19
Q

Which gel is acidic and which is alkaline?

A

Higher, stacking gel - slightly acidic

Lower, resolving gel - basic

20
Q

Which is more reliable, semi-dry or wet?

A

Wet

21
Q

What may explain unusual or unexpected bands in the results?

A

Protease degradation

22
Q

What may explain blurry bands in the results?

A

High voltage or air bubbles in transfer

23
Q

Why may no bands appear?

A

Improper antibody
Low concentration of antigen
Prolonged washing
Contamination

24
Q

What may lead to patchy/uneven spots on the blot?

A

Air bubbles

25
Q

Which neurotoxin do the PAGE gels contains?

A

Acrylamide

26
Q

Through which type of bonding do proteins bind to nitrocellulose?

A

Hydrophobic interaction

27
Q

What are the 3 components in stage 3 of western blotting?

A

Blocking
Antibody incubation
Detection

28
Q

What is the “blocking” component of protein detection?

A

Incubation of the blot with a protein or detergent

To prevent nonspecific binding of the antibodies to the membrane

29
Q

What is used as a blocking agent in Western blot?

A

Non fat dried milk

30
Q

Describe the antibody incubation of protein detection

A

Labelled primary antibody introduced to the blot

This binds to the protein on the nitrocellulose membrane

31
Q

What primary antibodies can be used as “house-keeping” or control?

A

anti-GAPDH

Actin

32
Q

Describe the final part of protein detection

A

Secondary antibody introduced which binds to the primary
This oxidises luminol to ground state in the presence of H2O2 in the enhanced chemiluminescence (ECL) to give off visible light when developed

33
Q

How is the molecular weight of the protein quantified in WB?

A

Compared to other molecular weight markers on the side of the blot
Or
Densitometer can be used to scan the blot of film and use software to compare the signal bands

34
Q

What is the purpose of the housekeeping protein?

A

To convey that an equal amount of protein lysate was loaded onto the gels for the mock and the DNA
If bands are equal size/intensity = equal amount of protein

35
Q

Are smaller proteins positively or negatively charged?

A

Negative

36
Q

The higher the gel & percentage the higher/lower the size of the protein?

A

Lower, in general

37
Q

Describe how the transfer stack is arranged?

A 
Neg electrode
Sponge
Filter paper
Gel
Membrane
Filter paper
Sponge
Pos electrode
38
Q

What does electrophoresis let you separate proteins by?

A

Weight (kDa)

39
Q

Why were the samples lysed with BMCE and SDS?

A

Denature proteins to be linear

40
Q

Why was the protein sample put on a heat block?

A

Aid denaturation

41
Q

What is the purpose of the Ponceau S?

A

To help visualise the bands if they are there

42
Q

What kind of molecules is non-fat dried milk not good for?

A

Fluorescent antibodies

43
Q

What can be used instead of 5% Marvel as a blocking agent?

A

BSA

44
Q

Why was the secondary antibody an anti-rabbit antibody?

A

Because the primary was made in rabbit

BMSc - Labs (6 decks)

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