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Advanced Higher Biology Unit 1: Cells and Proteins > Laboratory techniques for biologists > Flashcards

Laboratory techniques for biologists Flashcards

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1
Q

Tools to measure quantities accurately.

A

Scales
Measuring cylinders
Pipettes
Auto-pipettes

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2
Q

Why are dilutions used?

A

So a measurable value can be obtained.

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3
Q

Explain a LINEAR dilution series.

A

A range of dilutions differing by an equal interval (e.g: 0.1, 0.2, 0.3…).
Each concentration is made individually.

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4
Q

Explain a LOG dilution series.

A

A range of dilutions differing by a constant proportion.
The subsequent solution is made from diluting the solution before.
Earlier measurement errors are compounded in later dilutions.

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5
Q

Explain some of the things which a colorimeter would measure.

A

Measures the: Concentration of a pigment in a solution
Turbidity of a liquid
Density of cells in a culture

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6
Q

How does a colorimeter work?

A

Measures how much light is absorbed by a sample on a small transparent cuvette.
A denser sample shows a greater degree of transmission.

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7
Q

Explain a method used for a colorimeter which acts as a control.

A

The machine is calibrated using a blank cuvette which contains the solvent only.

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8
Q

Common uses of a colorimeter.

A

Measure the density of a cell culture growing in a liquid broth.
Measure the concentration of betanin leaking from denatured beetroot cells.
Measure the rate of reaction of enzymes like dopa oxidase.
Quantify the results of an ELISA demonstration.

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9
Q

How would you determine the concentration of a substance which has an unknown substance?

A

Plot the absorbance reading for a series of known concentrations of a substance/culture.
The line produced on the graph can be used as a reference for unknown concentrations of the same substance/culture.

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10
Q

The term which allows you to identify an estimate of the concentration of a substance.

A

Interpolation.

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11
Q

What is a buffer?

A

Aqueous solutions that show very little variation in pH.

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12
Q

How would you control the pH of a solution?

A

Using a buffer.

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13
Q

Give an example(s) of buffers in mammalian blood.

A

Carbonic acid

Bicarbonate anions

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14
Q

What is centrifugation?

A

The method of separating materials in suspension according to their density.

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15
Q

Describe the process of centrifugation acting on a cell sample?

A

Various constituent cellular structures and molecules are separated and can be studied in isolation.

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16
Q

Describe the range in rpm which material is rotated at.

A

2000-120000 rpm.

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17
Q

Where is material placed when it undergoes centrifugation.

A

A centrifuge tube.

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18
Q

What causes the constituents to separate.

A

G-force.

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19
Q

What is the name of the material which forms at the bottom of the centrifuge tube created by the denser items in a sample.

A

A pellet.

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20
Q

What is the liquid fraction above the pellet in a centrifuge tube called?

A

A supernatant.

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21
Q

What are the typical uses for centrifugation?

A

Enzyme extraction from tissues

Study of electron transport chain activity(kill reaction)

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22
Q

Types of chromatography.

A

Paper Chromatography

Thin Layer Chromatography.

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23
Q

Describe the use of chromatography.

A

The separation of amino acids according to their solubility.

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24
Q

What results in different rates of movement of substances in chromatography.

A

Different relative affinities of materials for the solvent.

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25
Q

Which part of a chromatography set up is hydrophilic?

A

The paper.

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26
Q

Which part of a chromatography set up is hydrophobic?

A

The solvent.

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27
Q

What is affinity chromatography?

A

The technique used to separate a specific protein from a mixture.

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28
Q

How are antibodies/ligands specific to proteins immobilised?

A

Form in agarose gels and packed into a column.

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29
Q

Explain how specific proteins are separated from a mixture in affinity chromatography.

A

The protein mixture is poured through the column and only the protein interested in binding to the antibody/ligand binds.
Other proteins don’t bind and can be washed away.

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30
Q

How would you recover the target protein in affinity chromatography?

A

Wash the column with a buffer(which lowers the affinity of the protein to the ligand) or a solution containing free ligand.

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31
Q

How does protein electrophoresis work?

A

The process uses the current flowing through a buffer to separate proteins depending on their size and charge.

32
Q

Does the protein running through electrophoresis need to be non-denatured or denatured?

A

No.
Non-denatured = native
Denatured = non-native

33
Q

How would you denature a protein?

A

With the presence of heat or detergent.

34
Q

What happens when a protein is denatured.

A

The chain unfolds.

35
Q

What does unfolding a protein do to its charge?

A

Results in a linear protein with a uniform charge along its length.

36
Q

How does unfolding a protein affect its rate of migration?

A

Rate of migration depends only on size(rather than charge and size).

37
Q

What is an isoelectric point?

A

The pH at which a protein has an overall neutral charge.

38
Q

How does a pH higher or lower than the isoelectric point affect the charge of a protein.

A

Causes the surface of the protein to be charged making it soluble.

39
Q

How is a protein soluble?

A

Water molecules interact with the proteins charge keeping it suspended in solution.

40
Q

What happens when the charge of the protein is neutral?

A

A solid precipitate forms out of the solution.

41
Q

How can you separate multiple different proteins in a mixture.

A

Slowly change the pH of the solution so different proteins become precipitates and can be extracted at their
different isoelectric points.

42
Q

What are antibody techniques used for

A

Detection and identification of specific proteins

43
Q

What are antibodies

A

Y-shaped globular proteins that bind to antigens

44
Q

Where and why are antibodies produced

A

Produced by b-lymphocytes as an immune response

45
Q

Where are b-lymphocytes produced

A

Spleen/bone-marrow

46
Q

Describe polyclonal antibodies

A

Many different antibodies attacking the same antigen

47
Q

Describe monoclonal antibodies

A

A supply of identical antibodies that will bind to the same feature of an antigen

48
Q

How do b-lymphocytes divide in culture

A

They are fused with myeloma cells from an immortal cell line using PEG.

49
Q

What is the name given to fused b-lymphocyte/myeloma cells

A

hybridomas

50
Q

How is the an antibody extracted and purified

A

Centrifugation

51
Q

What are monoclonal antibodies used for

A

Diagnosis and detection of disease

52
Q

How do immunoassay techniques work

A

Reporter enzyme linked to a monoclonal antibody produces a coloured product when a specific protein binds

53
Q

What is fluorescent-labeled protein blotting

A

A technique for identifying specific proteins that have been separated using gel electrophoresis.

54
Q

Explain how fluorescent-labeled protein blotting works

A

Proteins are blotted onto a nitrocellulose membrane to record their positions easily, the filter is flooded with fluorescent-labelled monoclonal antibodies which detect and identify the different proteins.

55
Q

Define histochemistry

A

The study of tissues using stains and histochemistry

56
Q

What is Fluorescent-immunohistochemistry used for

A

To visualise the distribution of specific cellular components in living cells

57
Q

Describe the growth mediums in cell cultures

A

Grown in suspension in a liquid medium

Grown on/within a solid medium

58
Q

Describe an inoculum

A

Growing cells are added to a culture from a previous culture (cells can be enzymatically removed or an explant may be taken)

59
Q

What are aseptic techniques used for

A

To prevent contamination by using sterile materials and appropriate treatments of sources

60
Q

Give some examples of aseptic techniques

A

Pass the neck of a bottle through a bunsen, pass the loop through a bunsen

61
Q

What is a growth factor

A

A complex medium which allow rapid cell division.

Consists of proteins, salts, vitamins, glucose, maybe antibiotics.

62
Q

How can growth factors be added to a culture

A

Via an animal serum

63
Q

Describe the pathway of cell culture

A

Cells are removed by proteolytic enzymes which adhere to the surface of the flask.
They then divide and form a monolayer until the nutrients are used up.
To keep the culture alive they must be transfered to a culture with more growth factors.

64
Q

What is the hayflick limit

A

The amount of cell divisions a culture can withstand before it dies (about 60)

65
Q

What is unique about myeloma (cancer cells)

A

The Hayflick limit doesn’t exist and the cell lines are immortal

66
Q

Define ‘totipotent’

A

Plant cell that is able to differentiate into all cell types to form a new organism

67
Q

Define explant

A

Small pieces of plant tissue that can be placed on a solid medium

68
Q

What are growth regulators used for

A

To induce embryogenesis to generate new embryonic plants

69
Q

What are haemocytometers used for

A

To count cell density through a known volume of medium

70
Q

What is a total and viable cell count

A

Total - All cells

Viable - Living cells (found by applying a stain to distinguish living cells)

71
Q

Define genome

A

The complete set of DNA

72
Q

Define proteome

A

The entire set of proteins that can be expressed from the genome

73
Q

Why is the proteome larger than the genome

A

Alternative RNA splicing

Post-translational modification

74
Q

What does an amino acid consist of

A

A carbon with a hydrogen, a carboxylic acid group, an amine group and an R group

75
Q

What are the classes of R groups

A

Acidic (COOH (negatively charged))
Basic (NH2 (positively charged))
Polar (OH)
Hydrophobic (hydrocarbon)

Advanced Higher Biology Unit 1: Cells and Proteins (2 decks)

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