Lab 1 Pipettes and Microscopy o 1000 uL 1mL o To use micropipettor push to first stop 2 3mm deep let slowly come up wait one sec withdraw from sample Dispense by placing it against the wall depress to first stop wait one second then to the second stop o 1 ocular unit 10 microns 0 01 mm o Nucleus 7 10um plasma membrane 4 5nm mitochondria 1 3 um Lysosome 0 2 0 5 um electron microscope 100nm and greater o Cells are transparent so contrast is an issue Change this by staining the cell both living and fixed cells not very specific Can also change microscope optics bright field and phase contrast Phase contrast makes use of varying thickness and biochemical properties of various organelles that causes different refraction Passes slightly retarded light through annulus that further retards like by wavelength Out of phase light is recombined with in phase light producing a visible intensity difference No staining need better for live cells o Basic light microscope visible light beneath the specimen passes through condenser lens which focuses the light on the specimen on the stage Light then passes through the objective lens above specimen Next light passes through ocular lens which magnifies image Lab 2 Spectrophotometry solution o Quantify number of proteins determine concentration of substance in o Chromophore chemical compounds that absorb light and absorb light energy at particular wavelengths based on their electronic structures Consist of conjugated pi systems or metal complexes Reagent dye usually contains chromophore not the protein Protein shifts or increase abs at particular wavelength o Light absorbance is directly proportional to its concentration OD ecl equivalent to A ebc o Bradford Assay binding CBBG 250 CBBG exits in red cationic 470nm form acidic conditions and when bound to proteins it becomes deprotonated and becomes blue detected at 595 nm More blue abs more protein o Standards can t find out much from just absorbance need to know concentration also Standard is sample of known concentration Need to have enough to span full range of likely protein s Need standard curve absorbance vs concentration and unknowns o If sample is too concentrated perform dilutions Look up in appendix of lab Lab 3 Physical Properties of Proteins and Methods of Isolation o Whole blood after centrifugation o Top 55 plasma 91 water 7 proteins 2 nutrients electrolytes hormones o Middle 1 buffy coat white blood cells platelets o Bottom 44 red blood cells most dense o Tissue Fractionation Tissue homogenization mechanical disruption so cells can be isolated These cells can then be lysed and components can be fractioned differentially Differential centrifugation spins at various speeds producing a centrifugal force much faster than gravity speeding the process of sedimentation The rpm that it separates at is based off size mass density Used this method for blood o Fractional Precipitation Exploits differential solubility can do this through change of pH addition of salt or an organic solvent We used last 2 Salting out NH4 2SO4 strip water molecules from shells proteins form aggregates and precipitate Changing dielectric constant add water miscible organic solvent like ethanol dielectric constant changes polarity higher polarity higher e ethanol lowers e lowers solvation protein protein interactions become stronger precipitates o SDS Page Sodium dodecysulfate polyacrylamide gel electrophoresis Charged molecules migrate in an electric field move based off size o Estimated protein concentration from standard curve x dilution factor actual protein concentration o Yield actual protein concentration x original volume of fraction o Ez fv E strengh of the electric field volts cm Z net charge on the molecule F frictional coefficient depends on size shape V velocity Velocity of a migrating chargd particle through an electric field is directly proportional to both the net charge on the molecule and strength of the field and inversely proportional to frictional coefficient o Detergent SDS binds to proteins proportionally to their masses heat unravels the protein so SDS can coat it unified charge to mass reducing agents may also be added to reduce disulfide bridges like DTT o PAGE gel matrix o Coomassie Blue stains gel Lab 4 Enzyme Kinetics o Structure of enzymes Active site small groove notch determined by tertiary structure Prosthetic group non protein at active site coenzyme organic cofactor metal ion o Enzyme control mechanisms Covalent modification phosphorylation or cleavage of an inactive precursor called a zymogen Enterokinase cleaves tripsinogen Environmental conditions pH temperature or ionic strength can affect delicate tertiary structure Compartmentalization must be carefully controlled so that metabolic rxns occur in the right place time prevent destruction of cell Contain in certain departments Production of inhibitors trypsin inhibitor prevents autocatalytic activation of trypsin in pancreas Trypsin serine protease zymogen called trypsinogen in pancreas Cleaved by enterokinase into its active form Trypsin can then activate itself autocatalytic o D L BAPA artificial substrate contains arginine residue after cleavage yields pNA rate of yellow pNA formation shows activity o Michaelis Menten Plots enzymatic rate as a function of substrate Km michaelis constant about same as dissociation constant 0 5Vmax value is substrate Vmax rate when enzyme is fully occupied with substrate Vo how do you find this Initial slope in concentration product vs time Two general types of inhibitors Competitive competes with substrate for spot in active site lowers Km but does not affect Vmax Noncompetitive binds to protein at another spot away from active site decreases ability to bind to enzyme decrease Vmax does not alter Km Lab 5 Membrane Receptors o Cell Communication Steps 1 Binding of extracellular ligand and receptor 2 Signal transduction relay of signal 3 Response o Autonomic Nervous system involuntary Sympathetic stimulates its targets increases heart rate Parasympathetic inhibits targets decreases heart rate o Agonist vs Antagonist Agonist binds to a receptor and triggers a response mimc action of naturally occurring substance Antagonist does not provoke a biological response upon binding to a receptor but dampens agonist mediated responses o Basic Daphnia features type of organism basal heart rate Fresh water crustacean aka water flea b c of hopping motion provide food Enough similarities Basal heart rate 300
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