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What are the two methods of protein detection?
Genetic approaches and Biochemical approach
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What do genetic approaches involve?
involve mutating the protein, and then looking at how the mutation effects cellular physiology
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What do the biochemical approaches involve?
isolate protein, what function does it perform in a test tube (enzymology involves a lot of this
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Give 3 examples of how eukaryotic cells' compartments each have their own set of proteins
-machinery for transcribing DNA into RNA needs to be in the nucleus -respiratory chain proteins in mitochondria -proteins involved in degrading macromolecules are contained in lysosomes.
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How many different proteins are then in a) E.coli, b) S.cerevisae c)Humans?
a)4800 b)6000 c)30 000-60 000
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What are antibodies secreted by?
B- cells
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How many different antibodies can the human body produce?
10^10
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How is this possible with only 30 000 genes?
because B cells and T cells can actually rearrange their DNA into different protein coding sequences
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What is monoclonal antibody?
Antibodies produced by a cloned hybridoma cell, which are therefore identical and directed against the same epitope of the antigen
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What are polyclonal antibodies?
A heterogeneous pool of antibodies produced by a number of different B-lymphocytes in response to an antigen. Different antibodies in the pool may recognise different epitopes on the antigen.
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What is an epitope?
Region of antigen to which antibody binds (can be linear or conformational)
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What can antibodies be tagged with?
- a fluorescent dye which can be detected using fluorescence microscopy - an enzyme that will cause a substrate to change colour - colloidal gold (electron dense) that can easily be seen under an electron microscope.
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Why is the secondary antibody tagged rather than the primary?
amplifies the signal and ensures that there is no change to the affinity of the primary antibody.
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What are the four ways we can break the cells open to access extracts/
Mild detergent (holes in membrane), sonication (high frequency sound), homogenisation (rotating plunger in glass tube), french press (cells forced through tiny hole at high pressure)
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What are the 3 types of centrifugation?
Differential (spin at increasing speeds- larger pellets out first), equilibrium (separates according to bouyant density) and velocity (sediments at different rates)
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What does SDS page stand for?
Sodium Dodecyl Sulphate- PolyAcrylamide Gel Electophoresis
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What is western blotting also known as?
Immunoblotting
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What are other methods of protein purification?
Fluorescent fusion proteins, sequence analysis, bioinformatics
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What are anabolic reactions?
Reactions responsible for growth and repair porcesses
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What are catabolic reactions?
Those that release energy needed to drive anbolic reactions
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What is ATP used as?
The energy currency of the cell, transfering energy captured during cellular respiration to cellular sites that use energy
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Where is ATPase found?
In the inner mitochondrial membrane, the chloroplast thlakoid membrane and the inner membrane of eubacteria
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What is chemiosmotic coupling?
The linkage of electron transport, proton pumping and ATP synthesis. In mitochondria this is known as oxidative phosphorylation.
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Name two agents that interfere with oxidative phosphorylation.
a) cyanide b) carbon monoxide.
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How do they work?
by inhibiting cytochrome oxidase, therefore blocking the passage of electrons to O2 and ATP synthesis grinds to a halt.
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What does one turn of the citric acid cycle produce?
3 NADH, 1 FADH2, 1GTP and releases 2 molecules of CO2
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What complex is used in the water splitting enzyme?
Special manganese splitting complex
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Why are the photosystems named as so?
Due to order of discovery
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Describe the three electron carriers.
 Plastoquinone (closely resembles ubiquinone of mitochondria)- Q  Plastocyanin (a small copper containing protein)- pC  Ferredoxin (a small protein containing an iron-sulphur centre)- Fd
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What are the ATP and NADPH created in light dependent reactions used for?
As energy sources in the fixation of carbon
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Where did mitochondria and chloroplasts orignate from?
The engulfment of bacteria by ancestral eukaryotic cells
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What is the dependence of the eukaryotic cell and the bacterial cell on eachother known as?
Endosymbiotic relationship
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What evidence is there for these origins?
Double membrane, small circular genomes, method of division (fission), occurence of DNA transcription, translation and replication in matrix/stroma
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What is the cytoskeleton?
A highly dynamic network of protein filaments throughout the cytoplasm/
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What is it important for?
Important for supporting a large volume of cytosol and shaping cell movement
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What is the role of intermediate filaments?
They provide tensile strength for cells.
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Where are they found?
anchored at the plasma membrane at cell junctions. They surround the nucleus and extend out to cell periphery. They are particularly abundant in cells that are subject to mechanical stress such as muscle cells, epithelial cells.
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Where are keratin filaments found?
Spanning the interior of epithelial cells
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What is a cadherin?
transmembrane proteins that span the bilayer and interact with ‘plaque’ proteins on the cytosolic side of membranes
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What are nuclear lamins?
A network of intermediate filaments under nuclear membrane
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What are laminins?
extracellular matrix proteins and NOT cytoskeletal proteins
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What are vimentins?
Intermediate filament found in connective tissue, muscle cells and neuroglial cells
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What are neurofilaments?
Intermediate filaments found in nerve cells
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Where is actin found?
in all eukaryotic cells
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In what case are actin proteins unstable?
Without associated proteins
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What are filopodia?
(finger/needle like projections of the plasma membrane)
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What is Lammelipodia?
(sheet like projections of the plasma membrane)
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How many myosin genes do humans have?
40
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What type of myosin is most abundant in the muscle?
II
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In what direction do most myosins move on the actin molecule. What is the exeption?
From - to +. The exception is myosin VI, which moves from + to -
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Describe the structure of microtubules
 Long hollow cylinders made up of tubulin monomers  More rigid and straight than intermediate or actin filaments  Typically grow from a microtubule organising centre such as centrosomes in animal cells  Have structural polarity
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Describe a tubulin monomer
dimer of alpha and beta tubulin held together by strong (but non-covalent) interactions
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How many proteins does a eukaryotic cell contain?
10 billion
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Give an example of the improtance of protein sorting in eukaryotes
Many (not all) enzymes that break down macromolecules are sorted and trafficked to lysosomes. The lysosomal membrane separates these degradative enzymes from the cytoplasm.
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Where does protein synthesis occur?
Nearly all occurs in the cytoplasm- except for mitochondria and chloroplasts which ahve their own protein synthesis machineries. However majority of their proteins are synthesised in the cytoplasm and must be imported to organelles.
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What is Ran-GAP
Ran-GTPase Activating Protein
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Whereabouts can mitochondrial proteins be found?
In the outer membrane, inner membrane, intermembrane space or matrix.
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Why is mitochondrial protein transport particularly complex?
 Mitochondria also have their own genome, and protein synthesis machinery.
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Card 2

Front

What do genetic approaches involve?

Back

involve mutating the protein, and then looking at how the mutation effects cellular physiology

Card 3

Front

What do the biochemical approaches involve?

Back

Preview of the front of card 3

Card 4

Front

Give 3 examples of how eukaryotic cells' compartments each have their own set of proteins

Back

Preview of the front of card 4

Card 5

Front

How many different proteins are then in a) E.coli, b) S.cerevisae c)Humans?

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Preview of the front of card 5
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