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Sociology 2152A/B
Doug Hazlewood

Cellular stress responses help ensure the cells survival or for elimination of damagedunwanted cells Whether the stress is environmental physiological or pathological the evolutionarily conserved response is to induce heat shock proteins HSPsHeat shock proteins act as molecular chaperones to help proteins fold properly they protect cells from extreme stressful conditions HSPs can also inhibit apoptotic pathways by interacting with cytoskeleton to maintain cell integrity Theyre grouped based on molecular weights HSP10 2030 and 100interacts with actin microfilaments of cytoskeletonHSP90constitutively expressed intracellular prevents premature folding of nascent polypeptides and interacts with microtubules of cytoskeleton HSP2770normal low basal levels increases in response to environmentalphysiological stress transcription is activated by heat shock factors HSF HSP70 can interact with microtubules microfilaments and intermediate filaments of the cytoskeletonHSF1master regulator in vertebrates constitutively expressed monomeric form interacts with HSP90 in the cytoplasm to stay inactive With stress amount of unfolded protein increases and competes with HSF1 for binding to HSP90 so HSF1 is released to form homotrimers that go to the nucleus to bind the promoter of HSP genes for transcription Heat shock stresshyperthermia will change protein expression and alter the cytoskeleton we need to extract and quantify proteins and stain to view the cytoskeleton MT MF IF Positive controlscell cultures treated with cytochalasin B microfilaments or colchicine microtubules show us the appearance of damaged but intact cytoskeleton systemsNegative controlcell culture without any treatment Heat shockinduced changes reversible Identify proteins sensitive to heat shock visualize cytoskeleton monitor effect of thermal stress on morphology of cytoskeletal systems and explain the responseTo extract proteins lyse cells destroy cell membranes with nonionic detergent Triton X100 or Nonident P40 and SDS in lysis buffer And to prevent endogenous proteases from degrading the proteins add protease inhibits EDTA leupeptin PMSF to the lysis buffer EDTA inhibits metalloproteases leupeptin inhibits serine and cysteine proteases and PMSF inhibits serine proteases RIPA buffer RadioImmuno Precipitation Assaylyses cultured mammalian cells extracts are compatible for protein assays immunoassays and protein purifications PAGE PAGEgel made by freeradical polymerization of acylamide and NNmethylenebisacrylamide free radicals from chemical decomposition of ammonim persulfate and stabilizes and catalyzed by TEMED Molecules move through set pore sizes based on polyacrylamide concentration charge size shape SDSamphipathic molecule that binds to hydrophobic parts of proteins to denature them often combined with heating and reducing agents DTT or mercaptoethanol to break disulfide bonds Glycerol make sample denser and minimize diffusion during loading
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