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| 10658801201 | proteonomics | A field of Biochemistry that determines what proteins are functionally relevant in a specific biological context. | 0 | |
| 10658804765 | genome | list of all genes and non-coding sequences of an organism | 1 | |
| 10658810080 | proteonome | list of all protein expressed by genome. | 2 | |
| 10658826411 | protein purification | Isolated protein is required to elucidate function. | 3 | |
| 10658831512 | differential centrifugation | Break cell membrane and centrifuge at high rate of speed *depending on force (speed) of centrifuge certain proteins will fractionate into the pellet |  | 4 |
| 10658835616 | needed to determine what fraction has protein of interest | assay of protein activity | 5 | |
| 10658841587 | Salting Out (Purification) | Proteins precipitate ("salt out") of solution at different [salt] |  | 6 |
| 10658845908 | dialysis | technique used to remove salt out of solution after salting out has occurred |  | 7 |
| 10658922799 | chromatography | purification by passing proteins in a mobile (aqueous) phase through a stationary (solid) phase | 8 | |
| 10658926309 | gel-filtration | separates proteins based on size -porous beads made of agarose or dextran interact with small molecules to slow their movement down the column *large molecules fractionate off column first |  | 9 |
| 10658929345 | ion exchange | separates proteins by their net charge -negative columns: separate out (+) charged molecules * elute desired proteins off with NaCl. Na+ will bind to negative column and displace (+) charged molecule |  | 10 |
| 10658932259 | affinity chromatography | pass mobile phase over column containing beads attractive to protein of interest |  | 11 |
| 10658935822 | recombinant proteins | affinity tag added to protein via recombinant DNA techniques | 12 | |
| 10658940475 | What attaches to protein and binds nickel(II) columns? | histidine tag | 13 | |
| 10658945812 | High-Pressure Liquid Chromatography (HPLC) | Significant pressure is applied to more mobile phase over more columns with very small beads • provides both higher resolution and faster separation -cost 2-15K + 400/column |  | 14 |
| 10658958790 | electrophoresis | separates a molecule with a net charge by passing an electric field through it. Molecule goes through a medium of unison viscosity, therefore, smaller molecules travel fastest slows movement of larger nucleotides | 15 | |
| 10658961856 | agarose | used to separate DNA and RNA molecules in linear form -DNA has a (-) charge due to phosphate backbone -0.75-1% agarose gel separates larger fragments 3-10Kb (kilobases) -1-2% Agarose gel separates shorter fragments 200bp to 3Kb *DNA must be linear or large coiled DNA will move quickly through the Gel | 16 | |
| 10658965602 | Polyacrylamide Gel Electrophoresis (PAGE): | Used to separate protein or very small nucleotide fragments; the porous gel slows the movement of larger proteins |  | 17 |
| 10659077096 | Sodium dodecyl Sulfate (SDS) | Ionic detergent used to denature protein by disrupting non-covalent interactions -binds proteins in uniform fashion: 1 SDS per 2 A.A. -Give protein large (-) charge in which charge is directly proportional to size of molecule | 18 | |
| 10659084630 | SDS-PAGE | used for protein separation and analysis of protein purification scheme -protein identified by Coomassie blue staining or Autoradiography (if radioactive label has been incorporated into the protein) | 19 | |
| 10659088624 | isoelectric focusing | separates on basis of acidic and base A.A. | 20 | |
| 10659094070 | isoelectric point | the pH at which the protein's net charge is 0 *electrophoresis is dependant on net charge for mobility, so when net charge is 0 protein stops -proteins are run on a gel with a pH gradient | 21 | |
| 10659097175 | 2D electrophoresis | used for very high resolution in separating protein *Combines SDS-PAGE with Isoelectric focusing. -the top gel separates proteins by pH via isoelectric focusing -the second gel separates proteins by size via SDS-PAGE *Allows you to further separate out different proteins of the same size | 22 | |
| 10659101121 | immunoglobin (Ig) | (aka antibody) a protein with a very specific and high affinity for a foreign substance called an Antigen -Antigens can be other proteins, polysaccharides, nucleic acids, peptides | 23 | |
| 10659107625 | epitope | specific functional group or A.A. group on antigen recognized by antibody | 24 | |
| 10659111258 | polyclonal | Heterogeneous mixture, meaning a mixture of antibodies that recognize the same antigen but at different epitopes | 25 | |
| 10659113477 | How are polyclonal antibodies produced? | 1. Injecting mouse or rabbit or rat with antigen (protein) of interest 2x about 3 weeks apart 2. animal creates antibodies against foreign antigen 3. bleed animal and centrifuge to separate blood cells from serum called Antiserum 4. Antiserum used as polyclonal antibody | 26 | |
| 10659119385 | monoclonal | Homogeneous mixture of identical antibodies recognizing one epitope -requires use of a cell line that will produce antibodies without dying outside of the host animal. Cells lines from the cancer multiple myeloma are used | 27 | |
| 10659122775 | How are monoclonal antibodies produced? | 1. injecting host animal with antigen of interest in similar fashion as polyclonal antibodies 2. spleen cells are isolated and its plasma is fused in vitro with myeloma cells 3. hybrid cells (Hybridoma cells) are screened for hybrid of choice 4. cell line that produces only the antibody of interest produces monoclonal antibodies | 28 | |
| 10659131588 | ELISA (enzyme-linked immunosorbent assay) | enzyme attached to antibody (typically monoclonal) will produce a color when substrate is added. Antibody will bind to antigen first, therefore color intensity is proportional to amount of antigen | 29 | |
| 10659138249 | indirect ELISA | antigen is used to detect antibody | 30 | |
| 10659143462 | HIV detection | person infected with HIV will create antibodies to viral protein | 31 | |
| 10659146953 | sandwich ELISA | antibody used to detect antigen; first antibody to antigen added doesn't have enzyme attached; second antibody to antigen added has enzyme attached |  | 32 |
| 10659151753 | drug screening | blood or urine added to microtiter plate with antibody attached to well. If antigen (drug) is present ELISA will detect it | 33 | |
| 10659157294 | Western Blot | incorporates gel electrophoresis to detect proteins 1. Run protein mixture on Gel Electro 2. transfer (blot) proteins to sheet 3. rinse sheet with antibody 4. place second antibody specific to first antibody - second antibody specific to first antibody - second antibody is labeled 5. view second antibody -if table is radioactive perform autoradiograph -if label is florescent perform immunofluorescence -detection of hepatitis C is performed via Western Blot |  | 34 |
| 10659692351 | fluorescence microscopy | allows study of proteins in vivo therefore one can understand the function/destination of a protein within the cell 1. fluorescently tag protein green florescent protein: (GFP) commonly used tag from jellyfish 2. watch protein movement in environmental change i.e. addition of hormone, change in temp etc. | 35 | |
| 10659707573 | mass spectrometry | sends molecule ions through gas phase and detects time it takes for molecule to pass F=ma F=force m=mass a=acceleration If F is constant then acceleration measurements can determine mass -small mass will pass through first | 36 | |
| 10659711191 | MALDI-TOF mass spectrometry | matrix-assisted laser desorption-ionization 1. Protein is placed on a matrix of crystallized molecules 2. a laser is pulsed at the matrix and ionized forms of the protein or peptide are released -helps ionize the protein -matrix protects protein from harm due to the laser 3. Time of Flight (TOF) is measured -smallest molecules first 4. results are computationally compared with a database of protein sequences to identify protein -Can be done with mixture of proteins in order to identify proteins involved in a complex or under certain conditions | 37 | |
| 10659738065 | X-ray crystallography | determines 3D structure of protein at atomic level; often at 1 or 2 Å resolution • wave length of x-ray (1.54 Å) similar to length of covalent bond • proteins must be crystallized so that they are in a fixed orientation with an orderly repeating fashion -most difficult step is crystal formation of protein - 100's to 1000's of attempts at different [salt] are often required to find optimal crystallizing condition * x-rays passed through crystal will scatter or diffract -diffraction occurs via encounter with atoms * amplitude of diffracted wave is directly proportional to number of e- | 38 | |
| 10659741665 | NMR spectroscopy | nuclear magnetic resonance provides structure information of protein while in solution -not as high resolution and requires higher [] protein solution * some atomic isotopes create magnetic spin *if constant magnetic field is applied and varying electromagnetic radiation is pulsed, the magnetic nuclei will switch spin or resonate *charge in spin is detected * different chemical groups (i.e. CH3 vs. CH2) will resonate at different electromagnetic radiation frequencies * special relationship of different groups also resonate at different frequencies | 39 | |
| 10659746715 | NOESY NMR Spectroscopy | nuclear Overhauser effect identifies protons in close proximity. Interaction between nuclei is inversely proportional to the distance between them; detection up to 5 Å or less | 40 |
