Molecular Cell Biology Flashcards
Compartmentalisation purpose
Focus on job at hand Concentrate on fewer things Organise Separate Coordinate
In prokaryotic can only complete limited processes due to all occurring in cytoplasm
Protein trafficking / secretion
Protein made in cytosol
Need to go through membrane, usually in unfolded state
Always have targeting signal
Signal may be 1-20 aas long, which is 1degree signal that says it belongs in compartment X
On entering compartment, signal sequence usually gets cleaved off, then rest of protein folds and commences function
Protein secretion
Secretory pathway involves transfer through membranes and encapsulation of proteins in vesicles, these vesicles transport protein to appropriate organelle. Occurs in ER, Golgi,
Antibody structure
Defensive immune response molecules. Made by B-lymphocytes (wbc’s)
Generated against antigen
Ags elicit ab production are almost always proteins but can be DNA, RNA or lipids
Y shaped, two light polypeptide chains, two heavy linked by multiple disulphide bonds
Coded for by nuclear genes
Goes to ER for assembly then secreted through vesicles out of cell into blood stream
Variable region- specifically tailored to bind to the Ag (recognition element)
Constant region- Fc portion binds to Fc receptors on lymphocytes, killer cells, macrophages
Bind very tightly to their Ags with tight affinities and high Ka values.
Require v high or low pH or SDS with boiling to uncouple.
Ab binds to small portion of Ag molecule, usually a sequence of element or a structural feature of the antigen called an epitope
2 variable Ag binding regions so Ab can cross link 2 molecules of Ag
Antibody production
Purify protein of interest
Inject to animal
If animal mounts an immune response to foreign protein (makes Abs against it), immunisation has been successful
Administer booster shots of Ag
Collect blood, harvest Ab from plasma/ serum
This approach produces polyclonal Abs, the most common form of which is immunoglobulin G (IgG)- recognise multiple epitope (features) on surface of Ag.
Another technique uses tissue culture to fuse B cell and myeloma cell to produce monoclonal Abs - each molecule of a mAb recognises a single epitope on the surface of Ag, so uniquely specific but more difficult, more expensive and slower to make
Immunogold electron microscopy (protein localisation technique that uses antibodies)
Standard electron microscopy EM reveals ultrastructure in thin sections of cells
Fix tissue in paraffin wax
Cut block into slices (few nm thick)
Stain with heavy metal (eg uranyl acetate, binds mainly to membranes)
EM passes electron beam through sample
Electrons readily pass through most materials but are deflected back by heavy metal
Creates negative image (intensity of signal depends on different densities in different regions of sample)
If we have antibodies to a protein of interest, they can be incorporated into an immunogold EM technique to determine where that protein is found in the cell
Add Ab specific to protein of interest (binds to Ag)
Add compound called ProteinA gold. Protein A is a bacterial protein from S aureus that binds to the Fc constant portion of IgG. Here it is covalently linked to gold particles, very dense.
Protein A binds only where the Ab against protein of interest previously bound. Ab itself won’t stop electrons passing through, nor will protein A, but the gold particles deflect electrons and appear as black dots on image.
Immunogold Em pros and cons
Pros
Hi res images
Helpful in understanding localisation due to intuitive image output (cellular context)
Cons
Fiddle
Microscope expensive
Needs skilled operator with expertise
Cells heavily fixed (wax, heavy dehydration); no longer alive and may not look like original live cells.
Static images only see a thin section of cell (so multiple slices?)
Immunofluorescence (IF) reagents
Protein localisation technique that uses antibodies
Primary antibody- directed against protein of interest. Often developed in rabbits or mice, usually IgG. Many Abs not commercially available.
Secondary antibody- recognises 1degree Ab. Developed by immunising a different animal with antibodies from 1degree Ab species (different animal sees these as foreign proteins). Often developed in goats, so many generate anti-rabbit Ab antibody, (eg. A goat derived Ab that will recognise and bind to all rabbit derived Abs)
Secondary Ab is conjugated (covalently linked) to a fluorescent compound for detection
Many 2degree Abs now commercially available- multiple species permutations, multiple fluorescent tags.
Immunofluorescence (IF) steps
Protein localisation technique that uses antibodies
Grow cells in tissue culture
Often use adherent cell types, eg skin cells (fibroblasts from small skin biopsy) or hela cells (lymphoma cancer cell line)
Take small skin biopsy, add cells to suitable tissue culture growth medium in Petri dish.
Under suitable condition, cells will grow and attach themselves to A plates like to be in contact with each other (plasma membrane projections, extra cellular proteins) so stick to most surfaces including glass microscope covers lips.
Look like fried egg (largest organelle, nucleus bulges in middle, rest of cell spreads out)
Need to get 1ab against protein of interest inside cell so it can bind to Ag, then add 2Ab conjugate so it can bind to 1Ab and fluoresce so we can see it’s location.
To get Abs through membrane, first add cross linking agent (formaldehyde, small molecule that passes through plasma membrane and joins things together like glue) - locks most molecules and organelles in place covalently (frozen in time) - fixing the cells/ fixation; maintains cell infrastructure
Dissolve membrane by adding detergent to allow Ab access to cellular components/ use non-ionic detergent, not SDS
Cells not fixed so cellular infrastructure remains intact, and membranes now mused allowing Ab access, but cells are dead.
Add 1Ab which will bind to protein of interest
Wash away excess Ab
Add 2Ab with fluorescent molecule attached, it will bind to 1Ab.
Wash away excess Ab
Ready for analysis
Immunofluorescence pros and cons
Pros
Relatively easy to perform
Fixing of cells is not so harsh
All cell components fixed together, so 3-D aspect through cell is intact- more of a whole cell picture
Can add additional fluorescent compounds eg. Organelles dyes for nucleus, mitochondria etc
Cons
Cells are dead so no real life dynamic
Subcellular fractionation and Western Blog Analysis
Protein localisation techniques that utilise antibodies
Relies on
Differential centrifugation of cell/ tissue lysates/ homogenates to enrich organelle specific fractions m
- based on density/ mass differences between different organelles
- which is largest organelle? Will it require a relatively low or high g force to be sedimented into the pellet on centrifugation? Which is the next largest organelle etc?
Western transfer or western blotting
- SDS page of cell extracts western blotting probe with primary Ab, then secondary Ab enzyme conjugate
Subcellular fractionation / western transfer
Pros
Provide extra info about protein size
Provides extra info about potential post translational modification (eg cleavage by protease, presence of other isoforms (multiple bands) due to alternative splicing of introns/ exons etc.
Cons
Fairly cumbersome
May suffer from contamination between organellar fractions
Green fluorescent protein (GFP)
Reporter molecule
Need to clone the gene for the protein of interest and link it to the sequence of GFP so that they work as one entity
Removal of stop codon from sequence of interest, and make sure the sequence will be in frame, can be achieved by using restriction endonucleases or PCR. Insertion of gene then by REs
GFP - good reporter molecule
- small monomeric protein
- gold by itself into the active fluorescent form. Helix contains the chromophore (light absorbing fluorescent group) formed by modification of Ser-try-gly
- folds independently of the folding of protein to which it is attached- C and N termini are well exposed
- very stable, once folded it is difficult to denature and it is resistant to proteases
- no intrinsic targeting signal of its own, meaning that any targeting signal found in the protein fused to GFP will determine targeting, and direct the fusion protein to the appropriate region of the cell for that protein.
- potentially, protein of interest can be fused at N or C terminus of GFP or vice versa, some proteins have a targeting signal at either the N or C
- can be expressed in almost any cell type
- can be visualised in live cells- time lapse providing important new info about cellular processes
- green fluorescence emitted by GFP can be complemented with other fluorescent dyes and we can detect co localisation of GFP with fluorescent organelle dyes
- has been modified to come in different flavours for different applications
Nuclear pore complex
Lots of components
Fibre point out toward cytosol (engage with proteins entering the nucleus)
Basket shape points in to nucleus (molecular sieve or filter, protein gate, offers selectivity)
Central core has more protein components that undergo dynamic re-arrangements depending on the protein that comes through
Small proteins made in the cytosol, if soluble and monomeric can freely diffuse through nuclear pores.
Larger proteins cannot pass through passively, but can be actively transported through a mechanism that causes the nuclear pore complex to undergo rearrangement
Nuclear lamina (laminate A, B and C)
On the inner face of nuclear envelope
Protein mesh called nuclear lamina
Comprises cytoskeletal components called Lamins A, B and C
Pairs of long beloved align and interwine to form coiled coils
Lamins support nuclear envelope and helps to hold entire nucleus together as a stable organelle
