Lecture 8 notes
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This 2 page Class Notes was uploaded by Brenna Eisenberg on Tuesday October 4, 2016. The Class Notes belongs to 327 at Syracuse University taught by erdman in Fall 2016. Since its upload, it has received 2 views. For similar materials see Cell Biology in Biology at Syracuse University.
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Date Created: 10/04/16
Lecture 8 Tuesday, October 4, 20110:59 AM • Fractionation: biochemical approach ○ Break apart the cells and tissues by physical or chemical means. Ex- using detergents, ultrasound ○ End up with membrane bound organelles and molecules • Differential centrifugation-repeat the centrifugation at faster and faster speeds ○ Larger, heavier material forms a pellet at the bottom ○ Can isolate specific membrane compartments like lysosomes • Column chromatography- proteins are placed in a column and immersed in solvent. They slow down at different rates based on size, charge, ability to bind to certain chemical groups, and hydrophobicity ○ Molecules that interact with the column will take longer to come out • Gel filtration chromatography- can separate proteins based on their size and shape ○ The matrix is made up of beads that can be of a certain shape. Smaller molecules that can fit inside the bead take longer tofilter out • Gel electrophoresis- applies an electric field to a solution with proteins.The direction and speed that the proteins travel reflects their charge and size • Antibodies can be used to purify and detect proteins.Can attach gold particles • Blotting- ○ Southern blot- run DNA in a gel, transfer the DNA onto a membrane, use a probe, can visualize DNA bands. Named after EM southern ○ Northern blotting- for RNA ○ Western blotting/ immunoblotting- used for proteins. The antigen is separated, and the antibodies will bind to them, allowing the position of the antigen to be determined • The structure of a protein can be predicted by NMR (used for smaller molecules) , X-ray crystallography (can be used for bigger structures), and enzymatic methods • Mass spectrometry is used to determine protein identity and modification proteomics. Ex- MALDI-TOF. Purify protein of interest and associated proteins. Proteins are fragmented and sized to identify them. Can separate by pH with 2D gel electrophoresis. Then subject them to heat shock and watch if some of the spots get bigger (more of ○ the protein), some disappear (no protein) ○ Cut out a single band from the gel. Produce peptides by digesting with Trypsin, cuts at a specific sequence. Peptides have a different size in the spectrometer, gives you the mass of the fragments. Very detailed molecular weight. • NMR- can determine the structure from the spectra Genetics • Make mutants by altering gene sequences. • Gene regulatory sequences- expression changes • Protein coding sequence changes- cause gene product change • Post transcriptionalgene regulation by siRNA's- double stranded, can knock out or knock down genes • CRISPR-Cas9 technology- used to make mutants or control gene expression. Known as genome editing • Dominant gain of function proteins can encode a poison subunit or proteins that are always "on". This is one approach for using mutants. ○ Can look at their phenotypes of the mutants ○ Can look for suppressors of the mutant's effects • PCR- polymerase chain reaction Add primers and separate the DNA strands with heat. After a few cycles, there is an exponential increase of the target sequence you Lecture 8 Page 1 ○ Add primers and separate the DNA strands with heat. After a few cycles, there is an exponential increase of the target sequence you are trying to study ○ Screen for inherited diseases ○ Can be used in southern blot for "deep sequencing", can create DNA microarrays. ○ Northern blot- can sequence RNAs in the genome ○ Western blot- PCR not very useful. Some protein microarrays. • FISH- fluorescence in situ hybridization ○ Labels parts of chromosomes Pharmacology- • Agonists- activators • Antagonists- inhibitors • Ex- colchicine and taxol are inhibitors of microtubule dynamics MEMBRANE STRUCTURES • Membranes allow cells to control how and where they interact with their environment ○ They allow the cell to receive information, import and export molecules, and move and expand ○ Ex- neutrophil extravasation-- can squeeze through endothelial cells to get to sites of infection • Membranes allow storage, concentration,and organization of proteins and other molecules • Proteins "float" in the fluid lipid bilayer. 2 dimensional. • The lipid bilayer forms vesicles-- it is energetically favorable, so the hydrophobictails are not exposed to the water. • Major components of bilayer: ○ Cholesterol- amphipathic. Small polar head groups. Packs in the lipid tails--reduces permeabilityand stiffens the membrane. ○ phospholipids Lecture 8 Page 2
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