Flashcards in Dr Patel's Work Deck (74):
Who made the first microscope?
Antony Van Leeuwenhoek
Simplistically what are microtubules?
Hollow cylindrical tubules of alpha and beta tubulin
Simplistically what are the centrosomes?
Main microtubule organizing centre (MTOC)
How did we discern the structure of a cell during mitosis?
We used fixed cells, at various stages combined with light microscopy.
We used live cells and GFP in synchronous colonies such as Drosophila embyros
What are the main types of microscopy?
What light wave phenomenon enables us to see images under a light microscope?
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
What is the magnification of a light microscope dependent upon?
The objective lens and to a smaller extent the eyepiece lens. Total magnification=eyepiece x objective
What is the resolution of a light microscope dependent upon?
The objective lens and the wavelength of the light source
What is Abbe's Law?
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
What equation is the numerical aperture of the lens given by?
What does the numerical aperture of the lens signify?
The light collecting ability of the lens, it is dependent on the medium between the object and the lens
What is contrast?
The ratio of light to dark
How can we increase the contrast in light microscopy?
Phase contrast of DIC (magnify small phase differences)
What are the key components of a typical compound microscope?
Eyepiece, Objective, Condensor and Light source
Give an example of when antibodies are used to diagnose a disease
Rabies can be diagnosed as the antibody detects viral nucleoproteins in infected brain tissue creating dots in the positive sample
Give an example of when antibodies can be used to treat a disease
Herceptin can treat some cancers in which EGF is overexpressed by binding to the Her2 receptor, stopping the signalling cascade causing excessive growth
What are the components of the innate response of our immune system
What are the main cells involved in the adaptive immune response?
T-cells produce cytokines and lymphokines
B-cells produce antibodies
What are epitopes?
The regions which the antibodies detect
What is clonal expansion?
The production of polyclonal antibodies
Describe the structure of an antibody
Y - composed of 2 Fab regions and an Fc region. It is made of 2 light and 1 heavy chain
What is the complement system?
A series of proteins are present within the plasma which are responsible for helping antibodies neutralize pathogens
Which antibodies are present as hormones?
IgG, IgD, IgE
What antibody is present in secretion?
Briefly outline phagocytosis
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
Outline histamine secretion in mast cells
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
How can so many different antibodies be formed?
They are derived from 2 different genes therefore combinations create variety
Outline the process for generating a polyclonal antibody
Inject live host with antigen and adjuvant
Allow to clot
Remove serum and put through an affinity column
What is an affinity column?
An agarose bead matrix with Protein A/G attached
How does an affinity column work?
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
What does clonal selection theory state
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
Describe the process of producing monoclonal antibodies
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.
How does the HAT selective medium work?
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
Give the general principle behind flourescence
When a fluorophores becomes excited it absorbs light, then when it becomes de-excited it releases a slightly lower frequency of light.
Give an example of a commonly used fluorescent dye
What are the Alexa Fluor dyes?
A series of bright photostable fluorophores
How are Alexa Fluor dyes conjugated to antibodies?
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
What is mitotracker?
A small fluorescent dye that binds specifically to mitochondria
Name a small fluorescent dye that binds specifically to DNA
What are the main constituents of a fluorescent microscope?
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
Outline the process of direct immunofluorescence microscopy
1. Fix cells using formaldehyde
2. Permeabilize cells
3. Incubate with 1% w/v BSA
4. Add FITC-conjugated antibody -> Ab binds to antigen
What is the structure of GFP?
Beta barrel with an alpha helical loop in the centre
What is a commonly occurring sequence in GFP?
What is the aim of confocal laser scanning microscopy?
How does confocal laser scanning microscopy work?
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
Outline sample preparation for an electron microscope slide
1. Specimen fixed in gluteraldehyde
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
What are the advantages of cell culture?
Controlled physiochemical environment
Controlled and defined physiological conditions
Homogenity of cell types
What are the two type of cell culture?
Primary - derived directly from tissue these have a short lifespan
Cell Lines - derived from primary cells or tumours, they have a long lifespan
In primary cells what limits cell divisions?
What is a typical tissue culture medium made up of?
Misc e.g. glucose, streptomycin, whole serum
Give an example of an ionic detergent
Give an example of a non-ionic detergent
How can proteins be removed from cells?
How is a cell extract prepared for SDS-PAGE?
Treated with SDS which unfolds the long polypeptides and binds giving it a net negative charge
Mercaptoethanol breaks disulphide bonds
How can pore size be varied in an SDS gel?
The concentration of polyacrylamide
Name two common stains used in SDS PAGE
Coomassie and Silver stain
Outline the process of western blotting
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
Give examples of reporters used in western blotting
Outline the process of affinity chromatography
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
Outline the process of MALDI-TOF MS
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
Outline the process of immunoprecipitation
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
6. Protein X/Ab/A sepharose bead complex precipitates
7. Assay by activity of identify through SDS PAGE
What is an epitope tag and give an example
Short protein fused to the target protein using DNA recombinant technology e.g. Myc Tag detected with the anti-Myc antibody
Outline the Yeast 2-Hybrid system
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
What are two common reporter genes used in the yeast 2 hybrid system?
HIS3 - histidien deficient medium
lacZ - cells appear blue due to the activation of beta galactosidase
How thick is the lipid bilayer?
How can lipids move in the bilayer?
Tails can flex
In rare circumstances flip
What makes up the outer leaflet of the cell membrane?
What makes up the inner leaflet of the cell membrane?
What does flippase do?
Maintain cell membrane asymmetry by detecting phosphatidylserine and phosphatidylethanoamine in the outer leaflet and flipping them to the inner leaflet using ATP.
What does scramblase do?
Remove cell membrane asymmetry in a cell undergoing apoptosis
What are lipid rafts?
Small specialized areas in the outer leaflet where some lipids and proteins are concentrated, they are composed of sphingolipids, cholesterol and proteins.
What two experiments demonstrate that proteins can diffuse laterally in the membrane?
What prevents H+ piggybacking on water in aquaporins?
2 opposite NPA motifs form H bonds with the lone pair on O