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BS2092 > Dr Patel's Work > Flashcards

Dr Patel's Work Flashcards

1
Q

Who made the first microscope?

A

Antony Van Leeuwenhoek

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2
Q

Simplistically what are microtubules?

A

Hollow cylindrical tubules of alpha and beta tubulin

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3
Q

Simplistically what are the centrosomes?

A

Main microtubule organizing centre (MTOC)

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4
Q

How did we discern the structure of a cell during mitosis?

A

We used fixed cells, at various stages combined with light microscopy.
We used live cells and GFP in synchronous colonies such as Drosophila embyros

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5
Q

What are the main types of microscopy?

A

Light
Electron
Fluorescence

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6
Q

What light wave phenomenon enables us to see images under a light microscope?

A

Interference, so constructive interference makes the image appear brighter and this occurs when the waves are in phase. Destructive interference occurs when the waves are out-of phase and makes the object appear darker

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7
Q

What is the magnification of a light microscope dependent upon?

A

The objective lens and to a smaller extent the eyepiece lens. Total magnification=eyepiece x objective

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8
Q

What is the resolution of a light microscope dependent upon?

A

The objective lens and the wavelength of the light source

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9
Q

What is Abbe’s Law?

A

R=0.61(lambda)/nsin(theta)
Where r= limit of resolution, n=refractive index of medium separating the specimen from the objective, theta = half the angular width of the cone of light collected by the objective

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10
Q

What equation is the numerical aperture of the lens given by?

A

nsintheta

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11
Q

What does the numerical aperture of the lens signify?

A

The light collecting ability of the lens, it is dependent on the medium between the object and the lens

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12
Q

What is contrast?

A

The ratio of light to dark

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13
Q

How can we increase the contrast in light microscopy?

A

Staining
Phase contrast of DIC (magnify small phase differences)
Illumination

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14
Q

What are the key components of a typical compound microscope?

A

Eyepiece, Objective, Condensor and Light source

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15
Q

Give an example of when antibodies are used to diagnose a disease

A

Rabies can be diagnosed as the antibody detects viral nucleoproteins in infected brain tissue creating dots in the positive sample

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16
Q

Give an example of when antibodies can be used to treat a disease

A

Herceptin can treat some cancers in which EGF is overexpressed by binding to the Her2 receptor, stopping the signalling cascade causing excessive growth

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17
Q

What are the components of the innate response of our immune system

A

Physical Barriers
Chemical Barriers
Cellular Defence

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18
Q

What are the main cells involved in the adaptive immune response?

A

T-cells produce cytokines and lymphokines

B-cells produce antibodies

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19
Q

What are epitopes?

A

The regions which the antibodies detect

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20
Q

What is clonal expansion?

A

The production of polyclonal antibodies

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21
Q

Describe the structure of an antibody

A

Y - composed of 2 Fab regions and an Fc region. It is made of 2 light and 1 heavy chain

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22
Q

What is the complement system?

A

A series of proteins are present within the plasma which are responsible for helping antibodies neutralize pathogens

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23
Q

Which antibodies are present as hormones?

A

IgG, IgD, IgE

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24
Q

What antibody is present in secretion?

A

IgA

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25
Q

Briefly outline phagocytosis

A

IgG binds to the antigens of the pathogen
The Fc receptors on the white blood cell bind to the IgG.
The pathogen is engulfed and broken down my hydrolytic enzymes

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26
Q

Outline histamine secretion in mast cells

A

IgE binds to Fc receptors on mast cells
Causes Ca to rise upon pathogen binding to antibody
This causes exocytosis of histamine granules
Histamine stimulates muscle contraction and vasodilation

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27
Q

How can so many different antibodies be formed?

A

They are derived from 2 different genes therefore combinations create variety

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28
Q

Outline the process for generating a polyclonal antibody

A

Inject live host with antigen and adjuvant
Remove blood
Allow to clot
Remove serum and put through an affinity column

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29
Q

What is an affinity column?

A

An agarose bead matrix with Protein A/G attached

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30
Q

How does an affinity column work?

A

When the serum is poured through the antibodies bind to protein A or protein G as these are prokaryotic cell wall proteins. The elution fractions = immunoglobulins

31
Q

What does clonal selection theory state

A

That if an isolated beta cell was cloned we would get a monoclonal antibody BUT this is difficult as beta cells have a short life span, can’t isolate it currently

32
Q

Describe the process of producing monoclonal antibodies

A

Mouse is immunised with antigen x and B lymphocytes are extracted these are then fused with a mutant cell line of B lymphocytes by suspending them in culture with a fusing agent then placing on a HAT selection medium, to remove any non-hybridoma cells. It is then centrifuged and the supernatant tested for anti-x antibody and cells from the positive well is cultured at bout 1 cell per well. The cells are allowed to multiple and individual supernatants are testes. Positive clones provide a continuous source of X antibody.

33
Q

How does the HAT selective medium work?

A

It only allows growth of heterokaryons by aminopterin blocking de novo DNA synthesis and hypoxyanthine and thymidine providing the cells with the raw materials required to use the salvage pathway

34
Q

Give the general principle behind flourescence

A

When a fluorophores becomes excited it absorbs light, then when it becomes de-excited it releases a slightly lower frequency of light.

35
Q

Give an example of a commonly used fluorescent dye

A

Flourescein Isthiocyanate

36
Q

What are the Alexa Fluor dyes?

A

A series of bright photostable fluorophores

37
Q

How are Alexa Fluor dyes conjugated to antibodies?

A
  1. Purify antibodies using Protein A/G
  2. Removed buffer and replace with PBS by dialysis and put in a buffer free of ammonium ions
  3. Add sodium bicarbonate ions
  4. Add dye, spin for 1 hour then leave at 4C overnight
38
Q

What is mitotracker?

A

A small fluorescent dye that binds specifically to mitochondria

39
Q

Name a small fluorescent dye that binds specifically to DNA

A

DAPI

40
Q

What are the main constituents of a fluorescent microscope?

A

Light source
1st Barrier filter - lets through only blue light
Beam splitting mirror - reflects light below 510nm and transmits light above 510nm
Second barrier filter - cuts off unwanted fluorescent signals

41
Q

Outline the process of direct immunofluorescence microscopy

A
  1. Fix cells using formaldehyde
  2. Permeabilize cells
  3. Incubate with 1% w/v BSA
  4. Add FITC-conjugated antibody -> Ab binds to antigen
  5. Observe
42
Q

What is the structure of GFP?

A

Beta barrel with an alpha helical loop in the centre

43
Q

What is a commonly occurring sequence in GFP?

A

Serine-Tyrosine-Glycine

44
Q

What is the aim of confocal laser scanning microscopy?

A

Observe 3D

45
Q

How does confocal laser scanning microscopy work?

A

Confocal pinholes focus the laser on entry
Emitted fluorescent light from the in focus point is focussed at the pinhole and reaches the detector.
Emitted light from an out of focus point is largely excluded from the detected due to the confocal pinholes

46
Q

Outline sample preparation for an electron microscope slide

A
  1. Specimen fixed in gluteraldehyde
  2. Dried
  3. Embedded in a block of plastic resin about 50-100nm thick using a microtome
  4. Stained using heavy metals e.g. lead, uranium and osmium
  5. Sections are placed on a small copper grid for viewing
47
Q

What are the advantages of cell culture?

A

Controlled physiochemical environment
Controlled and defined physiological conditions
Homogenity of cell types

48
Q

What are the two type of cell culture?

A

Primary - derived directly from tissue these have a short lifespan
Cell Lines - derived from primary cells or tumours, they have a long lifespan

49
Q

In primary cells what limits cell divisions?

A

Contact inhibition

Telomere shortening

50
Q

What is a typical tissue culture medium made up of?

A

Amino acids
Vitamins
Salts
Misc e.g. glucose, streptomycin, whole serum

51
Q

Give an example of an ionic detergent

A

SDS

52
Q

Give an example of a non-ionic detergent

A

Triton X-100

53
Q

How can proteins be removed from cells?

A 
Ionic detergent
Non-ionic detergent
Homogenization
Sonication
Freeze-thawing
54
Q

How is a cell extract prepared for SDS-PAGE?

A

Treated with SDS which unfolds the long polypeptides and binds giving it a net negative charge
Mercaptoethanol breaks disulphide bonds

55
Q

How can pore size be varied in an SDS gel?

A

The concentration of polyacrylamide

56
Q

Name two common stains used in SDS PAGE

A

Coomassie and Silver stain

57
Q

Outline the process of western blotting

A
  1. Transfer onto nitrocellulose membrane from gel by passing a current through it for about an hour
  2. Incubate the nitrocellulose membrane with 1% BSA
  3. Add primary antibody
  4. Add secondary antibody conjugated to a reporter molecule
58
Q

Give examples of reporters used in western blotting

A

Horseradish perioxidase

Alkaline phosphatase

59
Q

Outline the process of affinity chromatography

A
  1. Fuse the gene for protein X to the gene encoding the affinity tag e.g. Glutathione S-transferase
  2. Add glutathione beads which the GSP will bind to
  3. Wash with the appropriate buffer in this case glutathione
  4. Purify the protein complex
60
Q

Outline the process of MALDI-TOF MS

A
  1. Protein sample is mixed with a low molecular weight, UV absorbing organic acid and dried onto a metal plate
  2. Sample vaporised and ionised
  3. Ions accelerated in an electrical field
  4. Laser detects the ions measuring their time of flight which is proportional to root of m/z
61
Q

Outline the process of immunoprecipitation

A
  1. Add antibody to cell extract
  2. Ab binds to target protein X
  3. Add Protein A sepharose beads
  4. Protein X/Protein A sepharose bead complex forms
  5. Centrifuge
  6. Protein X/Ab/A sepharose bead complex precipitates
  7. Assay by activity of identify through SDS PAGE
62
Q

What is an epitope tag and give an example

A

Short protein fused to the target protein using DNA recombinant technology e.g. Myc Tag detected with the anti-Myc antibody

63
Q

Outline the Yeast 2-Hybrid system

A

Normal function of GAL4 is to bind to the GAL1 UAS which activates GAL1.

  1. GAL1 UAS is bound to a reporter gene
  2. The two domains of GAL4 are physically separated into plasmids
  3. These plasmids form 2 fusion proteins:
    BAIT - Protein X and DNA binding domain
    Prey - cDNA and Transcription activation domain
  4. Both plasmids inserted into Saccharomyces cerevisiae
  5. If proteins interact reporter gene will be expressed
64
Q

What are two common reporter genes used in the yeast 2 hybrid system?

A

HIS3 - histidien deficient medium

lacZ - cells appear blue due to the activation of beta galactosidase

65
Q

How thick is the lipid bilayer?

A

5nm

66
Q

How can lipids move in the bilayer?

A

Diffuse laterally
Rotate
Tails can flex
In rare circumstances flip

67
Q

What makes up the outer leaflet of the cell membrane?

A

Sphingomyelin
Glycolipids
Phosphatidylcholine

68
Q

What makes up the inner leaflet of the cell membrane?

A

Phophatidylserine

Phosphatidylethanoamine

69
Q

What does flippase do?

A

Maintain cell membrane asymmetry by detecting phosphatidylserine and phosphatidylethanoamine in the outer leaflet and flipping them to the inner leaflet using ATP.

70
Q

What does scramblase do?

A

Remove cell membrane asymmetry in a cell undergoing apoptosis

71
Q

What are lipid rafts?

A

Small specialized areas in the outer leaflet where some lipids and proteins are concentrated, they are composed of sphingolipids, cholesterol and proteins.

72
Q

What two experiments demonstrate that proteins can diffuse laterally in the membrane?

A

FRAP

Cell fusion

73
Q

What prevents H+ piggybacking on water in aquaporins?

A

2 opposite NPA motifs form H bonds with the lone pair on O

74
Q

What is a carrier protein? Give an example

A

Protein that mediates passive transport of large uncharged polar molecules across the cell membrane e.g. GLUT4 transporter

BS2092 (5 decks)

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