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102 Cards in this Set
- Front
- Back
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What are the methods to avoid contamination?
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Autoclave, filtration, Irradiation.
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How does Autoclave work?
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It uses high pressure and temperature to kill microbes
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How does filtration work?
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It passes the solution through a membrane with a small pore size (.45 to .2 microns)
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What would be a good reason to use filtration or Irradiation instead of Autoclave?
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Because you do not want to boil everything, especially plastic
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what are the two aseptic or sterile technique!
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Lamina flow hood, and antubiotics
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What are the different types of antibiotics used today.
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Penicillin, streptomyocin, gentamicin, and antifungals
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How does penicillin work?
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By killing grand-positive bacteria
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How does streptomyocin work?
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It kills grand negative bacteria
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how does Gentamicin work?
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expensive, not used in the lab
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What are Antifungals?
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They are expensive and not used in the lab.
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Why are viruses able to get us dominate the cells and are immune to all these defense mechanism?
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Viruses go right through the filters and they are not affected by antibiotics. Microplasm also very resistant to methods to avoid contamination.
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Are microplasms resistant to most methods of contamination avoidance?
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Yes
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What are the two media most used for growing cells?
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DMEM and RPMI
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What's in DMEM? And what does it look like?
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sugar, salts, fenal read, bycarbs and it looks like cool aid
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What's in RPMI1640?
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sodium bycarbonate, inorganic salts, amino acids, vitamins, glucose.
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What's phenol red?
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it is a pH indicator. on 7.4pH it looks like cool aid. On purplish the pH is too high
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List supplements used in the growing of cells:
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hormones, growth factors, insulin, transferin (iron binding protein), fetal calf serum (lots of growth hormone, 5-10% of the medium for growing cells)
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What are the optimal supplements for growing cells?
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sodium pyruvate (so that the cells dont work to hard to produce energy), non-essential amino acids, L-glutamine, and Heppes buffer(very expensive, not contingent, not used in lab)
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What are the optimal growing conditions for cells?
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37 C, humid atmosphere, and proper CO2 content.
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What is the job of the incubator when growing cells?
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it maintains the temperature constant at 37C, maintains humidity, and it maintains the proper CO2 content for cells to grow at the best rate possible.
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What are the different types of tissue cell culture?
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Primary explants and continuous cell lines
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What are primary cell explants?
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cells derived directly from tissue or blood
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What are continuous cell lines?
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cell cultures of the same cell type
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What happens to continuous cell cultures after 12 weeks of growing?
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They transform because of mutations
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What are the types of cell lines?
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non-tranformed, transformed and cancer cell lines
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What are non-transformed cell lines?
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they are essentially normal and have a limited life span
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What are transformed cell lines?
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These cells are altered to show continuous growth
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What are cancer cell lines?
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They are derived from cancers. The show continual growth and may have altered functions
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Cell characteristics:
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non-adherent(leukocytes) , adherent (epithelial, fibroblast. they stick to the glass)`
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What is the growth pattern of epithelial cells?
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they usually grow flat and spread out
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Describe the growth patterns of fibroblasts and endothelial cells:
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Fibroblasts are connective tissue that grows in the form of a spindle. Endothelial cells line the blood vessels and the grow by spreading and growing flat(endothelial cells look like a fried egg)
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What are the cell types used in our lab?
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IEC-6 (rat small intestinal epithelial, non-transformed), CaCo-2 (human carcinoma, transformed), and 3T3(mouse fibroblast, transformed)
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What does it mean when two things are confluent?
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it means that the flow well together, they blend into one another.
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What is apoptosis?
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programmed cell death
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What is necrosis and why is it bad?
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necrosis is when cells die because of damage. Necrosis is bad because the cell swell then burst, releasing intracellular contents(enzymes, DNA, etc) all over and potentially damaging neighboring cells.
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Why is apoptosis good, as opposed to necrosis?
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because in apoptosis (programmed cell death), the cells die because of natural causes and they are recycled, avoiding the spillage of the cell's content
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Why do cells undergo apoptosis?
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we naturally rid cells that we do not need. Remove excess cells from the immune system, kill cells that are infected, and cells that are under stress because a lack of growth factors
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What is trypsin?
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is an enzyme that digests the proteins in the extracellular matrix (pig pancreas for example)
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What is EDTA?
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is a chelator (removes metals from the blood) that removes Ca and Mg, thus breaking the bonds between the integrins and the extracellular proteins
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What are Trypsin and EDTA used for?
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When combined trypsin and EDTA make a dynamic duo that remove the cells from the flask the adhere to
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What are the morphological changes in apoptosis?
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chromatic condenses, DNA fragments(200bp fragments), cytoskeleton and nuclear envelope disassemble, cells bleb and fragment and form apoptotic bodies.
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What do cells that bleb (apoptotic) look like?
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raspberries
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What signals the cells to begin cell division?
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it is a combination of factors. Hormones, growth factors, chemicals, and physical agents
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What is EGF?
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Epidermal growth factor is a growth factor that promotes the proliferation of cells. It is produced by a number of cell types and it affects different cell types (affected cells must have EGF receptors). It induces cells in G0 and G1 phase to enter the S phase. It has a major role in development and wound healing.
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What are the techniques used to measure cell proliferation?
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used the hemacytometer, tritiated thymidine uptake, measuring the enzimatic activity (MTT assay), and DNA binding dyes.
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What are the benefits and problems with using a hemacytometer to count the number of cells?
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the benefits are that you get the actual number of cells but it is a tedious process, its time consuming and it is impractical if you are using many samples.
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What are the benefits and problems with using tritiated thymine uptake?
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The benefits are that it is very sensitive, its effects are logarithmic, its adapted to be used with 96-well plates, and it doesnt have any RNA. The problems are that it is radioactive, and it contaminates all materials that come in contact with it. Thus, making its disposable waste hazardous and expensive
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How is tritiated thydimine uptake used to count cells?
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During the last 4-10hrs titriated (H3) thimidine is added. Cells use it to make nucleotides and the H3(radioactive) is incorporated into the newly synthesized DNA.
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How does MTT (Enzymatic activity) is used to count cells?
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MTT is added in the last 4 hrs. Mitochondrial dehydrogenases cleave MTT to form a purple product. The color is measure with a spectometer. The more purple, the more enzyme and therefore the more cells.
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What are the benefits and problems of using MTT to count cells?
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The benefits are that it is not radioactive, it can be adapted to 96-well plates and it is relatively simple. The problems are that its effects are not logarithmic, it is less sensitive, it is not a direct measure of cell number and MTT is a carcinogen.
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How do we use DNA binding dyes to count the number of cells?
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We added it to the culture and the dye enters the cell and binds to DNA. We wash out the excess and measure its fluorescence using the cytofluor II
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What are the benefits and problems with using DNA binding dyes to count the number of cells?
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The benefits are that it is similar to tritiated thidimine but it is not radioactive, it is simple to do, and cytofluor can read 96-24-12 well plates. The problems are that the binding dyes are potential mutagens and SYTO dyes are very expensive
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What are the techniques used in cell disruption?
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Sonification, Freeze, detergents, and proteases
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What is sonification?
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It is a technique used for cell disruption that uses high frequency sounds to move the cells
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How does freeze (cell disruption) work?
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Freeze work by thawing (gradual warming from frozen) the cells.
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What are detergents and how do they interact with the cells?
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Detergents are used to solubilize the cell membranes. they solubilize intergral membrane proteins.
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What some examples of detergents:
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Ionic: Sodium dodecyl sulfate (SDS), and sodium desoxycholate. (used when protein-protein interaction is not needed, the proteins are denatured)
Non-ionic: Triton X-100 and Nonidet P-40. (used when protein-protein interaction is needed) |
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What are proteases? And how do they work?
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Proteases sequester specific parts of the cell. Proteases can begin to destroy your sample proteins, to avoid this you must keep your sample on ice. The ice brings the samples down to a temperature where enzymes cannot work.
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How does EGF work in the 3T3 cells?
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It is working through kinases. Kinases phosphorylate proteins, turn on and off proteins and phosphorylate tyrosine amino acids and serine/theronine amino acids
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Is the EGF receptor a kinase? If so how does it work?
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Yes. The cytoplasmic tail phosphorylates intracellular signaling proteins at tyrosine amino acids. The tyrosine phosphorylation starts the signaling pathway. The signaling pathways eventually activates one or more transcription factors. T.F move to the nucleus and bind to gene promoter/enhancer region. This trunks on the gene to allow transcription of mRNA. Two genes can be activated by the same transcription factor.
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What are the percentages of acrylamide for the SDS PAGE gel?
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4% acrylamide for the stacking gel and 12% for the resolving gel.
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At what wavelength do proteins absorb light?
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280nm
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why is the Christian and Wanburg correction equation used?
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because DNA and RNA absorb light too.
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What is the Christian and Wanburg equeation?
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Protein = (A280) x (correction factor) x (dilution factor) x (mg/ml)
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What is SDS-PAGE?
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is a technique used to separate proteins based on their electrophoteric mobility.
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How does SDS work?
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It is a strong ionic detergent that quickly solubilizes the cell, and denature proteins. It linearizes the protein and it gives it a negative charge.
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Who was Laemmli?
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Laemmli is the most cited scientist in history. He is responsible for creating the discontinuous stacking bands effect (4% acrylamide stacking gel over 12% acrylamide resolving gel).
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What is the purpose of the SDS-PAGE gel?
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to separate proteins in a polyacrylimide matrix
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What is TEMED?
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tetramethylethylenediamide
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What is 2-mercaptoethanol?
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2-ME reduces disulfide (-s-s-) bonds. Skunks use it as a defense mechanism and it is also used to give cooking gas its particular smell.
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What's in the reducing buffer?
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2-ME, Tris-HCL, glycerol, and 0.5 % bromophenol blue.
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What is the function of glycerol in the reducing buffer?
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It gives weight to the sample so that it settles at the bottom of the well.
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What is Tris-HCL?
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a buffer to maintain pH
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What is 0.5% bromophenol blue?
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Its a small molecule that rides along with the protein solution to serve as a dye marker on the gel. It goes faster than the proteins.
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What techniques are used to identify unknown proteins?
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Wester blooting
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What is the function of nitrocellulose?
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It sticks to proteins (protein whore)
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Explain the procedure of wester blooting
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The proteins transfered into a nitrocellulose matrix. Then antibodies are used to identify the proteins
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How are western blot antibodies made?
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Antibodies are made from vertebrates (rabbit or goat) that bind antigens to foreign proteins. Purify the protein, inject it into the animal, bleed the animal after a month and purify the antibody from the blood. This antibodies are made by B-lymphocytes and they bind to the foreign proteins with high affinity and high specificity.
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What is the shape of antibodies?
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The have a Y shape and the have two identical binding sites.
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What is Coomassil Blue?
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It is a protein stain that it is not used in this lab.
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Antibodies are proteins too, because of this we have to avoid getting the antibodies stuck in the nitrocellulose matrix. How is this accomplished?
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We saturate the nitrocellulose by adding bovine serum. Thereafter, no other proteins can stick to the nitrocellulose matrix.
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In which ways can you label an antibody when using wester blots?
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by using either a fluorescent, radioactive or enzyme compound
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When using western blot, the secondary antibody has to be from a different species?
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Yes
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Cell lines may be ...
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adherent or non-adherent
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What is the passage number? What has to happen for the passage number to change? What is another name for passage number?
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Every time from a cell line we need to increment the passage number. The passage number indicates the amount of time the cells have been in culture since the primary explant. Passage number is also known as subculturing.
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What is Trypan Blue?
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is a vital dye that is actively secluded from life cells but the dead ones stain blue
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What are apoptotic bodies?
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are the form that cells take when undergoing apoptosis. This blebbing is caused by the condensation of the nuclear material, nuclear fragmentation and the cytoplams blebs into small packages of cellular material
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What are death receptors and name one or two.
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Death receptors are the receptors of the cell that signal cell death. Two of these receptors are the Fas receptor and the tumor necrosis receptor.
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What is the importance of apoptotic bodies?
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These are important because none of the extracellular material (DNA, Enzymes, etc) is released to the outside and no damage is done to the neighboring cells.
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Why is necrosis bad?
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Because in necrosis (cell death due to tissue damage) the cells swell and burst, releasing all the intracellular materials. This material can cause an inflamatory response and begin the process of inflamation
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What is SYTO 24 and how does it work?
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This is a colorless dye that freely passes through the cell membrane but binds with DNA and it becomes a fluorescent green compound. This dye is excited by a blue light at 485nm wavelength and it emits light at 530nm. This was the dye used to see the apoptotic bodies in the cells undergoing apoptosis. Because the DNA condenses in apoptotic cells a bright green color can be seen in cells undergoing apoptosis.
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What is the role of EGF?
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It stimulates cells to increase cell proliferation
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Before you use PAGE and Western Blots what do you need to do to the cells?
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Disrupt them to release the cell's proteins. This can be achieve by the used of detergents (SDS).
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What is the purpose of the Lowry assay?
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To determine the protein concentration in a sample. This assays was chosen over other ones because it is less affected by detergents (SDS) than the others. ELISA is used to determine the protein concentrations.
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What is the receptor for EGF? VERY INTUITIVE!!!!
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EGF-R
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What is spectrophotometry based on?
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It is based on the idea that different chemicals posses unique bonding characteristics and are comprised of different atoms. Thus, each chemical will interact with light in a different ways.
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The plastic polymer used in PAGE is...
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Acrylamide
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PAGE stands for...
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Polyacrilymide gel electrophoresys
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EGF induces...
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Tyrosine phosphorylation
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What are cytokines?
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small proteins that secreted by one cell can affect the function of itself or nearby cells
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What does it mean to have a synergistic effect?
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a synergistic effect almost doubles the effects put together
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How many time should you do an experiment to test for validity of the data?
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It depends on the level of significance you get, but the absolute minimum is 3 times (3 replicates of each experiment)
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