BIS 104 Lecture 3 PDF

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Professor Daniel Starr
Fall 2021 Cell biology Lectures
All notes for midterms and quizzes...

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BIS 104 Lecture 3 1. Immunoprecipitation a. Isolate a single protein or complex out of cell b. Need antibody against protein of interest c. Purify antibody protein complex i. Lyse cell: shear forces (breaks membranes) - Dounce homogenizer (break apart membrane without denature) ALSO CAN USE non-ionic detergent (don’t denature the protein) ii. Clear lysis - centrifugation insoluble proteins and aggregates pellet (supernatant is the cytosol) iii. Add antibody against protein of interest 1. Protein A from bacteria (what the antibody recognizes and binds to) bacteria can evade the immune system 2. Put protein A on a bead that will precipitate out (bind to complex and centrifuge) 2. Antibodies in Medicine a. Ex. herceptin used in breast cancer: some breast cancers 50,000/year in US called HER2+ i. HER 2+ = overexpression of HER2 protein which is a receptor normally activate cell to divide, but in breast cancer, there are a ton of HER 2 receptor so over proliferate ii. ONCOGENE BECAUSE ITS NORMAL FUNCTION TELL THE CELL TO DIVIDE iii. Herceptin is a “humanized” monoclonal antibody- won’t be rejected because “humanized” clinical antibodies (manmade with mice) 1. Humanize means it uses the human IgM bottom of Y shape of antibody and house IgM gene recognizes human HER2 top of Y iv. Herceptin binds and blocks HER 2 signaling and blocks signaling pathways to prevent division 3. Subcellular fractionation a. Separate organelles based on speed of sedimentation in centrifuge b. Velocity density gradients i. Lyse cells with Dounce homogenizer to maintain organelles 1. ER, Golgi, Plasma membrane break from vesicle microsomes 2. Low speed centrifugation at 600xgravity - cause nuclei to go to pellet, everything else go to supernatant 3. Take supernanent and do faster centrifugation at medium speed (15000xg)- larger organelles like mito, ribo, and lysosomes go to pellet a. Supernatant will have cytosol, microsomes, ER/Golgi,etc.

4. Centrifuge at a higher speed (100000xg), now cytosol will go to supernatant and microsomes (ER,Golgi, plasma membrane) will go to pellet 5. Separating membrane vesicles: centrifuge microsomes in a sucrose density gradient 4. Looking at cells microscopy a. Transmitted light microscopy- use condenser to focus light onto and through a specimen through the sample i. Objective lens to collect and magnify the light - resolve 2 things apart and use the distance 2 things are apart to see them = d ii. d= 0.61λ/ (nsin α) iii. .61 = constant iv. Λ = wavelength of light nm v. n= refractive index media between sample and lense air=1 and oil = 1.4 vi. α= angle of light that enters the objective vii. The best resolution of a light microscope is 200nm apart b. Fluorescence microscopy i. fluorophore= molecule when excited by one wavelength light and when it falls it emits a photon at a lower energy and has different colored light so longer wavelength 1. Rhodamine excited green 535nm and emit red light2. No movement anymore so not dynamic: so cannot watch protein in real time 3. To watch in real time: found gfp c. GFP= green fluorescent protein i. Aequorea victoria - jellyfish puget sound 1. Uv light -> gfp -> green light 2. Gene for gfp + fuse to gene of interest express fusion protein your gene + gfp then watch your protein in real time 3. Flouraflour helix type (not mentioned in lecture) shaped barrel 4. See cells divide

d. Electron Microscopy- use electrons instead of photons much smaller wavelength smaller d distance resolved 2 objects i. d=.3 nm 1. E- accelerated into a beam by high voltage = light like properties 2. Focus electrons with magnets 3. Problems: need to be in a vacuum and sample must be fixed (so snapshots not real time) a. Cryopreservation

b. Chemical cross linkers c. Electron beam is very damaging- start to degrade sample d. Sample needs to be very thin so electron beam can pass through itpass through different speeds and part to show contrasts e. Low contrasts= need to stain samples

1) Core concepts how to study cells e. Location of protein can describe the function i. To localized protein: differential centrifugation, gfp, immunofluorescence 2) Proteins rarely work alone: work in complexes and networks a) Immunoprecipitation 3)Cellular processes are dynamic b) Cyclical -> cell division, transport c) Plasticity -> ability to respond to multiple signals...