MCAT Lab Techniques Guide

IWantAHighMCATScore’s Guide​ ​to​ ​MCAT​ ​Lab​ ​Techniques Gel​ ​Electrophoresis Purpose:​ ​Separation​ ​of​ ​proteins,​ ​DNA,​ ​or​ ​RNA​ ​based​ ​on​ ​size​ ​and/or​ ​charge Macromolecules​ ​(proteins,​ ​DNA,​ ​or​ ​RNA)​ ​of​ ​interest​ ​are​ ​placed​ ​in​ ​the​ ​lanes​ ​of​ ​a​ ​gel.​ ​For​ ​pro...

IWantAHighMCATScore’s Guide​ ​to​ ​MCAT​ ​Lab​ ​Techniques Gel​ ​Electrophoresis Purpose:​ ​Separation​ ​of​ ​proteins,​ ​DNA,​ ​or​ ​RNA​ ​based​ ​on​ ​size​ ​and/or​ ​charge Macromolecules​ ​(proteins,​ ​DNA,​ ​or​ ​RNA)​ ​of​ ​interest​ ​are​ ​placed​ ​in​ ​the​ ​lanes​ ​of​ ​a​ ​gel.​ ​For​ ​proteins​ ​and small​ ​molecules​ ​of​ ​DNA​ ​and​ ​RNA,​ ​the​ ​gel​ ​will​ ​be​ ​polyacrylamide.​ ​For​ ​larger​ ​molecules​ ​of​ ​DNA​ ​(>​ ​500​ ​bp), the​ ​gel​ ​will​ ​be​ ​agarose.​ ​An​ ​electrical​ ​charge​ ​is​ ​placed​ ​across​ ​the​ ​gel.​ ​At​ ​the​ ​bottom​ ​is​ ​the​ ​positively charged​ ​anode​​ ​and​ ​at​ ​the​ ​top​ ​is​ ​the​ ​negatively​ ​charged​ ​cathode​.​ ​Keep​ ​in​ ​mind,​ ​since​ ​a​ ​voltage​ ​source​ ​is applied​ ​to​ ​gel​ ​electrophoresis,​ ​it​ ​follows​ ​the​ ​same​ ​principles​ ​as​ ​an​ ​electrolytic​ ​cell.​ ​Negatively​ ​charged molecules​ ​will​ ​travel​ ​toward​ ​the​ ​anode.​ ​Because​ ​of​ ​the​ ​resistance​ ​of​ ​the​ ​gel,​ ​larger​ ​molecules​ ​will​ ​have​ ​a harder​ ​time​ ​moving​ ​and​ ​thus,​ ​the​ ​molecules​ ​will​ ​be​ ​separated​ ​by​ ​size​ ​with​ ​the​ ​smallest​ ​molecules toward​ ​the​ ​bottom.​ ​The​ ​gel​ ​can​ ​then​ ​be​ ​stained​ ​for​ ​visualization,​ ​typically​ ​using​ ​Coomassie​ ​Blue​ ​dye.​ ​A lane​ ​will​ ​be​ ​loaded​ ​with​ ​a​ ​collection​ ​of​ ​molecules​ ​of​ ​a​ ​known​ ​size,​ ​called​ ​a​ ​ladder,​ ​which​ ​can​ ​used​ ​to determine​ ​the​ ​size​ ​of​ ​the​ ​molecules​ ​being​ ​ran.​ ​There​ ​are​ ​several​ ​different​ ​applications​ ​of​ ​gel electrophoresis: Native-PAGE Native-PAGE​ ​is​ ​a​ ​polyacrylamide​ ​gel​ ​electrophoresis​ ​method​ ​for​ ​proteins​ ​that​ ​occurs​ ​under non-denaturing​ ​conditions.​ ​This​ ​method​ ​will​ ​separate​ ​proteins​ ​by​ ​size​ ​while​ ​retaining​ ​their​ ​structure. SDS-PAGE SDS-PAGE​ ​is​ ​a​ ​polyacrylamide​ ​gel electrophoresis​ ​method​ ​for​ ​proteins​ ​that occurs​ ​under​ ​denaturing​ ​conditions​ ​to separate​ ​proteins​ ​by​ ​mass.​ ​Negatively-charged sodium​ ​dodecyl​ ​sulfate​ ​(SDS)​ ​is​ ​added​ ​to​ ​the​ ​solution​ ​of​ ​proteins,​ ​denatures​ ​the​ ​proteins,​ ​and​ ​binds​ ​one SDS​ ​for​ ​every​ ​two​ ​amino​ ​acids,​ ​giving​ ​all​ ​proteins​ ​the​ ​same​ ​charge-to-mass​ ​ratio.​ ​Since​ ​all​ ​proteins​ ​have the​ ​same​ ​charge-to-mass​ ​ratio,​ ​they​ ​are​ ​separated​ ​solely​ ​on​ ​mass,​ ​with​ ​the​ ​smallest​ ​proteins​ ​found toward​ ​the​ ​bottom​ ​of​ ​the​ ​gel.​ ​SDS​ ​will​ ​only​ ​interrupt​ ​non-covalent​ ​bonds,​ ​so​ ​if​ ​disulfide​ ​bridges​ ​are present​ ​in​ ​the​ ​protein,​ ​they​ ​will​ ​not​ ​be​ ​broken.​ ​This​ ​is​ ​useful​ ​when​ ​analyzing​ ​proteins​ ​with​ ​multiple subunits. Reducing​ ​SDS-PAGE Reducing​ ​SDS-PAGE​ ​is​ ​exactly​ ​like​ ​SDS-PAGE,​ ​but​ ​with​ ​the​ ​addition​ ​of​ ​a​ ​reducing​ ​agent,​ ​like β-mercaptoethanol,​ ​which​ ​will​ ​reduce​ ​disulfide​ ​bridges​ ​and​ ​result​ ​in​ ​a​ ​completely​ ​denatured​ ​protein. Isoelectric​ ​Focusing Isoelectric​ ​focusing​ ​is​ ​a​ ​gel electrophoresis​ ​method​ ​that​ ​separates proteins​ ​on​ ​the​ ​basis​ ​of​ ​their​ ​relative contents​ ​of​ ​acidic​ ​and​ ​basic​ ​residues.​ ​A polyacrylamide​ ​gel​ ​with​ ​a​ ​pH​ ​gradient (low​ ​pH​ ​on​ ​one​ ​side,​ ​high​ ​pH​ ​on​ ​the other)​ ​is​ ​used.​ ​When​ ​proteins​ ​migrate through​ ​the​ ​pH​ ​gradient​ ​gel,​ ​they​ ​will travel​ ​toward​ ​the​ ​anode​ ​until​ ​the​ ​area​ ​of the​ ​gel​ ​with​ ​the​ ​pH​ ​that​ ​matches​ ​their isoelectric​ ​point​ ​(pI).​ ​When​ ​a​ ​protein​ ​is​ ​at​ ​its​ ​pI,​ ​it​ ​has​ ​a​ ​net​ ​charge​ ​of​ ​zero​ ​and​ ​will​ ​not​ ​be​ ​attracted​ ​to the​ ​positively​ ​charged​ ​anode​ ​so​ ​it​ ​will​ ​not​ ​move. Western​ ​Blotting​ ​(Protein) Purpose:​​ ​Detection​ ​of​ ​a​ ​specific​ ​protein​ ​in​ ​a​ ​sample Step​ ​1:​​ ​Proteins​ ​from​ ​a​ ​sample​ ​are​ ​loaded​ ​into​ ​an​ ​SDS-PAGE​ ​gel​ ​and​ ​separated​ ​based​ ​on​ ​size Step​ ​2:​ ​Proteins​ ​from​ ​the​ ​gel​ ​are​ ​transferred​ ​to​ ​a​ ​polymer​ ​sheet​ ​and​ ​exposed​ ​to​ ​a​ ​radiolabeled​ ​antibody (sometimes​ ​using​ ​two​ ​antibodies;​ ​one​ ​specific​ ​to​ ​the​ ​protein​ ​of​ ​interest​ ​and​ ​another​ ​radiolabeled antibody​ ​that​ ​binds​ ​to​ ​the​ ​first​ ​antibody)​ ​that​ ​is​ ​specific​ ​to​ ​our​ ​protein​ ​of​ ​interest Step​ ​3:​ ​The​ ​polymer​ ​sheet​ ​is​ ​viewed​ ​used​ ​autoradiography.​ ​The​ ​protein​ ​of​ ​interest​ ​that​ ​is​ ​bound​ ​to​ ​the radiolabeled​ ​antibody​ ​will​ ​be​ ​visible. Southern​ ​(DNA)​ ​and​ ​Northern​ ​(RNA)​ ​Blotting Purpose:​ ​Detection​ ​of​ ​a​ ​specific​ ​DNA​ ​(Southern​ ​blot)​ ​or​ ​RNA​ ​(Northern​ ​blot)​ ​sequence​ ​in​ ​a​ ​sample Step​ ​1:​ ​The​ ​DNA​ ​strand​ ​of​ ​interest​ ​is​ ​exposed​ ​to​ ​restriction​ ​enzymes​ ​that​ ​cut​ ​the​ ​DNA​ ​strand​ ​into smaller​ ​fragments Step​ ​2:​​ ​ ​The​ ​newly​ ​cleaved​ ​strands​ ​of​ ​DNA​ ​are​ ​denatured​ ​using​ ​a​ ​solution​ ​of​ ​NaOH​ ​to​ ​create​ ​ssDNA strands Step​ ​3:​​ ​The​ ​single​ ​stranded​ ​cleaved​ ​strands​ ​of​ ​DNA​ ​undergo​ ​gel​ ​electrophoresis,​ ​separating​ ​them​ ​by size.​ ​Smaller​ ​fragments​ ​will​ ​be​ ​found​ ​at​ ​the​ ​bottom​ ​of​ ​the​ ​gel.​ ​Polyacrylamide​ ​is​ ​used​ ​if​ ​the​ ​stands​ ​are less​ ​than​ ​500​ ​base​ ​pairs.​ ​Agarose​ ​is​ ​used​ ​if​ ​the​ ​strands​ ​are​ ​over​ ​500​ ​base​ ​pairs. Step​ ​4:​ ​The​ ​DNA​ ​from​ ​the​ ​gel​ ​is​ ​transferred​ ​to​ ​a​ ​sheet​ ​of​ ​nitrocellulose​ ​paper​ ​and​ ​then​ ​exposed​ ​to​ ​a​ 32​ ​ P radiolabeled​ ​DNA​ ​probe​ ​that​ ​is​ ​complementary​ ​to​ ​our​ ​DNA​ ​of​ ​interest. Step​ ​5:​ ​The​ ​nitrocellulose​ ​paper​ ​is​ ​then​ ​viewed​ ​using​ ​autoradiography​ ​to​ ​identify​ ​the​ ​strand​ ​of​ ​interest. NOTE:​ ​These​ ​methods​ ​are​ ​nearly​ ​identical​ ​for​ ​Southern​ ​and​ ​Northern​ ​blotting.​ ​The​ ​steps​ ​listed​ ​above are​ ​for​ ​Southern​ ​blotting,​ ​however,​ ​the​ ​only​ ​difference​ ​is​ ​that​ ​Northern​ ​blotting​ ​uses​ ​RNA,​ ​so​ ​steps​ ​1 and​ ​2​ ​are​ ​not​ ​done. DNA​ ​Sequencing​ ​(Sanger​ ​Dideoxynucleotide​ ​Sequencing) Purpose:​​ ​Used​ ​to​ ​determine​ ​the​ ​sequence​ ​of​ ​nucleotides​ ​in​ ​a​ ​strand​ ​of​ ​DNA Modified​ ​nucleotides,​ ​known​ ​as​ ​“dideoxynucleotides”​ ​(ddNTPs),​ ​are​ ​used​ ​in​ ​this​ ​method.​ ​ddNTPs​ ​are missing​ ​the​ ​OH​ ​group​ ​on​ ​the​ ​3’​ ​carbon,​ ​thus​ ​they​ ​are​ ​unable​ ​to​ ​create​ ​a​ ​new​ ​5’→3’​ ​phosphodiester bond.​ ​This​ ​allows​ ​us​ ​to​ ​control​ ​the​ ​termination​ ​of​ ​replication. Step​ ​1:​​ ​The​ ​DNA​ ​strand​ ​of​ ​interest​ ​is​ ​denatured​ ​using​ ​an​ ​NaOH​ ​solution​ ​to​ ​create​ ​a​ ​ssDNA​ ​strand​ ​that we​ ​can​ ​use​ ​for​ ​replication Step​ ​2:​​ ​The​ ​ssDNA​ ​strand​ ​of​ ​interest​ ​is​ ​added​ ​to​ ​a​ ​solution​ ​containing: 1. A​ ​radiolabeled​ ​DNA​ ​primer​ ​that is​ ​complementary​ ​to​ ​the​ ​gene​ ​of interest 2. DNA​ ​polymerase 3. All​ ​four​ ​dNTPs​ ​(dATP,​ ​dTTP, dCTP,​ ​dGTP) 4. A​ ​very​ ​small​ ​quantity​ ​of​ ​a​ ​single ddNTP​ ​(e.g.,​ ​ddATP) This​ ​step​ ​is​ ​done​ ​once​ ​for​ ​each​ ​of​ ​the four​ ​nucleotides​ ​in​ ​separate​ ​solutions. Step​ ​3:​ ​Each​ ​solution​ ​containing​ ​a specific​ ​dNTP​ ​and​ ​ddNTP​ ​are​ ​placed​ ​in their​ ​own​ ​lane​ ​of​ ​a​ ​gel​ ​and​ ​ran​ ​under​ ​gel electrophoresis.​ ​The​ ​gel​ ​is​ ​transferred​ ​to a​ ​polymer​ ​sheet​ ​and​ ​autoradiography​ ​is used​ ​to​ ​identify​ ​the​ ​strands​ ​in​ ​the​ ​gel. For​ ​each​ ​respective​ ​nucleotide,​ ​the​ ​insertion​ ​of​ ​a​ ​ddNTP​ ​will​ ​terminate​ ​replication​ ​and​ ​create​ ​various strands​ ​of​ ​different​ ​length​ ​that​ ​correspond​ ​to​ ​that​ ​specific​ ​nucleotide.​ ​Therefore,​ ​the​ ​gel​ ​can​ ​be​ ​read from​ ​bottom-to-top​ ​to​ ​determine​ ​the​ ​nucleotide​ ​sequence.​ ​The​ ​smaller​ ​the​ ​fragment,​ ​the​ ​further​ ​it travels​ ​in​ ​the​ ​gel. Chromatography Purpose:​ ​The​ ​separation​ ​of​ ​two​ ​or​ ​more​ ​molecules​ ​from​ ​a​ ​mixture There​ ​are​ ​several​ ​different​ ​types​ ​of​ ​chromatography​ ​that​ ​can​ ​be​ ​used​ ​for​ ​separating​ ​or​ ​analyzing​ ​a mixture​ ​of​ ​two​ ​or​ ​more​ ​molecules​ ​based​ ​on​ ​their​ ​properties.​ ​Traditionally,​ ​there​ ​are​ ​two​ ​components​ ​to chromatography;​ ​a​ ​stationary​ ​phase​,​ ​which​ ​is​ ​typically​ ​polar,​ ​and​ ​a​ ​mobile​ ​phase​,​ ​which​ ​is​ ​typically non-polar.​ ​Polar​ ​molecules​ ​are​ ​separated​ ​from​ ​a​ ​mixture​ ​by​ ​staying​ ​with​ ​the​ ​stationary​ ​phase,​ ​while non-polar​ ​molecules​ ​stay​ ​with​ ​the​ ​mobile​ ​phase.​ ​However,​ ​if​ ​reverse-phase​​ ​is​ ​specified,​ ​then​ ​the properties​ ​of​ ​the​ ​two​ ​phases​ ​are​ ​switched.​ ​This​ ​also​ ​changes​ ​based​ ​on​ ​the​ ​type​ ​of​ ​chromatography used,​ ​as​ ​some​ ​methods​ ​use​ ​ligands​ ​or​ ​gel​ ​beads. Liquid​ ​Chromatography In​ ​liquid​ ​chromatography,​ ​silica​ ​is​ ​traditionally​ ​used​ ​as​ ​the​ ​stationary​ ​phase while​ ​toluene​ ​or​ ​another​ ​non-polar​ ​liquid​ ​is​ ​used​ ​as​ ​the​ ​mobile​ ​phase. High-Performance​ ​Liquid​ ​Chromatography​ ​(HPLC) HPLC​ ​is​ ​a​ ​type​ ​of​ ​liquid​ ​chromatography​ ​that​ ​utilizes​ ​high​ ​pressures​ ​to​ ​pass the​ ​solvent​ ​phase​ ​through​ ​a​ ​more​ ​finely-ground​ ​stationary​ ​phase,​ ​which increases​ ​the​ ​interactions​ ​between​ ​the​ ​molecules​ ​and​ ​the​ ​stationary​ ​phase, giving​ ​HPLC​ ​a​ ​higher​ ​resolving​ ​power.​ ​Molecules​ ​can​ ​then​ ​be​ ​determined based​ ​on​ ​their​ ​absorbance​ ​and​ ​elution​ ​time​ ​as​ ​seen​ ​on​ ​the​ ​right. Gas​ ​Chromatography Gas​ ​chromatography​ ​(also​ ​known​ ​as​ ​gas-liquid​ ​chromatography)​ ​is​ ​used​ ​to​ ​separate​ ​and​ ​analyze molecules​ ​that​ ​can​ ​be​ ​vaporized.​ ​The​ ​mobile​ ​phase​ ​is​ ​an​ ​inert​ ​or​ ​unreactive​ ​gas,​ ​such​ ​as​ ​helium​ ​or nitrogen,​ ​while​ ​the​ ​stationary​ ​phase​ ​is​ ​a​ ​thin​ ​layer​ ​of​ ​liquid​ ​or​ ​polymer​ ​that​ ​surrounds​ ​the​ ​walls​ ​of​ ​a tube.​ ​The​ ​stationary​ ​phase​ ​allows​ ​more​ ​polar​ ​molecules​ ​to​ ​elute​ ​slower,​ ​giving​ ​them​ ​a​ ​higher​ ​retention time. Gel-Filtration​ ​(Size​ ​Exclusion)​ ​Chromatography Gel-filtration​ ​chromatography​ ​(also​ ​known​ ​as​ ​size-exclusion chromatography)​ ​is​ ​used​ ​to​ ​separate​ ​molecules​ ​by​ ​size​ ​rather​ ​than polarity.​ ​Smaller​ ​molecules​ ​can​ ​enter​ ​the​ ​porous​ ​gel​ ​beads,​ ​allowing​ ​them to​ ​elute​ ​later,​ ​while​ ​larger​ ​molecules​ ​that​ ​do​ ​not​ ​fit​ ​will​ ​elute​ ​faster.​ ​The gel​ ​beads​ ​can​ ​be​ ​viewed​ ​as​ ​the​ ​stationary​ ​phase,​ ​while​ ​the​ ​solution​ ​in​ ​the column​ ​can​ ​be​ ​viewed​ ​as​ ​the​ ​mobile​ ​phase. Ion-Exchange​ ​Chromatography Ion-exchange​ ​chromatography​ ​will​ ​separate​ ​proteins​ ​by​ ​their​ ​net​ ​charge.​ ​The column​ ​is​ ​filled​ ​with​ ​charged​ ​beads,​ ​either​ ​positive​ ​or​ ​negative.​ ​In anion-exchange,​ ​negatively-charged​ ​beads​ ​are​ ​used​ ​which​ ​attract​ ​positively charged​ ​proteins​ ​and​ ​negatively-charged​ ​proteins​ ​will​ ​elute​ ​first.​ ​In cation-exchange,​ ​positively-charged​ ​beads​ ​are​ ​used​ ​which​ ​attract negatively-charged​ ​proteins​ ​and​ ​positively-charged​ ​proteins​ ​will​ ​elute​ ​first. Affinity​ ​Chromatography Affinity​ ​chromatography​ ​will​ ​separate​ ​proteins​ ​based​ ​on​ ​their affinity​ ​for​ ​a​ ​specific​ ​ligand.​ ​Beads​ ​that​ ​are​ ​bound​ ​to​ ​a​ ​specific ligand​ ​will​ ​be​ ​used​ ​and​ ​proteins​ ​with​ ​a​ ​high​ ​affinity​ ​for​ ​that​ ​ligand will​ ​bind​ ​to​ ​the​ ​beads,​ ​allowing​ ​proteins​ ​with​ ​a​ ​low​ ​affinity​ ​to​ ​elute first.​ ​The​ ​high​ ​affinity​ ​proteins​ ​are​ ​then​ ​eluted​ ​by​ ​increasing​ ​the concentration​ ​of​ ​the​ ​free​ ​ligand​ ​in​ ​the​ ​column,​ ​which​ ​competes​ ​for the​ ​active​ ​site​ ​of​ ​the​ ​bound​ ​proteins. Thin-Layer​ ​Chromatography Thin-layer​ ​chromatography​ ​consists​ ​of​ ​a small​ ​sheet​ ​of​ ​medium​ ​that​ ​is​ ​coated​ ​in​ ​an adsorbent​ ​material,​ ​such​ ​as​ ​silica​ ​gel.​ ​The polar​ ​silica​ ​is​ ​the​ ​stationary​ ​phase.​ ​The molecules​ ​of​ ​interest​ ​are​ ​added​ ​to​ ​the bottom​ ​of​ ​the​ ​sheet​ ​and​ ​the​ ​sheet​ ​is​ ​placed in​ ​a​ ​non-polar​ ​liquid,​ ​such​ ​as​ ​heptane,​ ​until​ ​it reaches​ ​the​ ​origin.​ ​The​ ​mobile​ ​phase​ ​then travels​ ​up​ ​the​ ​plate​ ​using​ ​capillary​ ​action, allowing​ ​the​ ​molecules​ ​to​ ​move​ ​with​ ​it​ ​if they​ ​are​ ​relatively​ ​non-polar.​ ​The​ ​spots​ ​are then​ ​visualized​ ​using​ ​UV​ ​light. The​ ​relative​ ​distances​ ​traveled​ ​between​ ​the molecules​ ​is​ ​represented​ ​by​ ​the​ ​R​f​​ ​value,​ ​which​ ​is​ ​measured​ ​as​ ​the​ ​ratio​ ​of​ ​the​ ​distance​ ​the​ ​molecule traveled​ ​from​ ​the​ ​origin​ ​to​ ​the​ ​distance​ ​the​ ​solvent​ ​front​ ​traveled​ ​from​ ​the​ ​origin. Distillation Purpose:​ ​Used​ ​to​ ​separate​ ​two​ ​or​ ​more​ ​molecules​ ​from​ ​a​ ​solution Simple​ ​Distillation Simple​ ​distillation​ ​is​ ​used​ ​to​ ​separate​ ​two​ ​molecules​ ​from​ ​a​ ​solution​ ​when​ ​their​ ​boiling​ ​points​ ​differ​ ​by 25​o​​ ​C​ ​or​ ​greater. Fractional​ ​Distillation Fractional​ ​distillation​ ​is​ ​used​ ​to​ ​separate​ ​two​ ​molecules​ ​from​ ​a​ ​solution​ ​when​ ​their​ ​boiling​ ​points​ ​differ by​ ​less​ ​than​ ​25​o​​ ​C. Vacuum​ ​Distillation Vacuum​ ​distillation​ ​is​ ​used​ ​to​ ​separate​ ​two​ ​molecules​ ​from​ ​a​ ​solution​ ​when​ ​their​ ​boiling​ ​points​ ​are​ ​high and​ ​risk​ ​changing​ ​chemically. Polymerase​ ​Chain​ ​Reaction Purpose:​​ ​Used​ ​to​ ​amplify​ ​a​ ​small​ ​quantity​ ​of​ ​DNA​ ​by​ ​several​ ​orders of​ ​magnitude Step​ ​1:​​ ​DNA​ ​strands​ ​and​ ​complementary​ ​DNA​ ​primers​ ​are​ ​heated to​ ​95​o​​ ​C​ ​for​ ​15​ ​seconds​ ​to​ ​separate​ ​the​ ​strands. Step​ ​2:​​ ​The​ ​solution​ ​is​ ​abruptly​ ​cooled​ ​to​ ​54​o​​ ​C​ ​to​ ​allow​ ​the​ ​primers to​ ​anneal​ ​to​ ​each​ ​ssDNA. Step​ ​3:​ ​The​ ​solution​ ​is​ ​heated​ ​to​ ​72​o​​ ​C​ ​and​ ​new​ ​complementary strands​ ​are​ ​synthesized​ ​using​ ​Taq​ ​DNA​ ​polymerase. Step​ ​4:​​ ​The​ ​cycle​ ​is​ ​repeated​ ​until​ ​the​ ​desired​ ​quantity​ ​of​ ​DNA​ ​is synthesized. Spectroscopy Purpose:​ ​Used​ ​for​ ​structural​ ​determination​ ​of​ ​molecules H-NMR​ ​Spectroscopy 1​ The​ ​application​ ​of​ ​nuclear​ ​magnetic resonance​ ​regarding​ ​the​ 1​​ H​ ​isotope​ ​within the​ ​molecules​ ​of​ ​a​ ​substance. Chemical​ ​shift​:​ ​The​ ​chemical​ ​shift​ ​on​ ​the x-axis​ ​(δppm)​ ​represents​ ​the​ ​amount​ ​of deshielding​ ​of​ ​electrons​ ​that​ ​is​ ​caused​ ​by an​ ​adjacent​ ​heteroatom​ ​or​ ​pi​ ​bond. - 0​ ​-​ ​5​ ​ppm​ ​→​ ​Alkane​ ​region - 3​ ​-​ ​5​ ​ppm​ ​→​ ​Alkane​ ​with​ ​a heteroatom​ ​region - 5​ ​-​ ​7​ ​ppm​ ​→​ ​Alkene​ ​region - 6​ ​-​ ​8​ ​ppm​ ​→​ ​Aromatic​ ​region - 9​ ​-​ ​10​ ​ppm​ ​→​ ​Aldehyde​ ​region - 10​ ​-​ ​13​ ​ppm​ ​→​ ​Carboxylic​ ​acid region Integration:​ ​The​ ​integration​ ​of​ ​the​ ​peak​ ​determines​ ​the​ ​number​ ​of​ ​equivalent​ ​hydrogens​ ​a​ ​signal represents. Neighbors:​ ​The​ ​number​ ​of​ ​peaks​ ​determines​ ​the​ ​number​ ​of​ ​neighboring​ ​hydrogens​ ​that​ ​are​ ​≤​ ​3​ ​bonds away.​ ​The​ ​number​ ​of​ ​peaks​ ​equals​ ​the​ ​number​ ​of​ ​neighbors​ ​+​ ​1. - Singlet​ ​→​ ​No​ ​neighboring​ ​hydrogens - Doublet​ ​→​ ​One​ ​neighboring​ ​hydrogen - Triplet​ ​→​ ​Two​ ​neighboring​ ​hydrogens - Quartet​ ​→​ ​Three​ ​neighboring​ ​hydrogens - Quintet​ ​→​ ​Four​ ​neighboring​ ​hydrogens - Sextet​ ​→​ ​Five​ ​neighboring​ ​hydrogens - Septet​ ​→​ ​Six​ ​neighboring​ ​hydrogens - Multiplet​ ​→​ ​Seven​ ​or​ ​more​ ​neighboring​ ​hydrogens C-NMR​ ​spectroscopy 13​ The​ ​application​ ​of​ ​nuclear​ ​magnetic​ ​resonance​ ​regarding​ ​the​ 13​ ​ C​ ​isotope​ ​within​ ​the​ ​molecules​ ​of​ ​a substance Chemical​ ​shift​:​ ​The​ ​chemical​ ​shift​ ​on​ ​the​ ​x-axis​ ​(δppm)​ ​represents​ ​the​ ​amount​ ​of​ ​deshielding​ ​of electrons​ ​that​ ​is​ ​caused​ ​by​ ​an​ ​adjacent​ ​heteroatom​ ​or​ ​pi​ ​bond. - 0​ ​-​ ​70​ ​ppm​ ​→​ ​Alkane​ ​region - 90​ ​-​ ​120​ ​ppm​ ​→​ ​Alkene​ ​region - 110​ ​-​ ​160​ ​ppm​ ​→​ ​Aromatic​ ​region - 160​ ​-​ ​200​ ​ppm​ ​→​ ​Carbonyl​ ​region IR​ ​Spectroscopy IR​ ​spectroscopy​ ​is​ ​a​ ​method​ ​that​ ​is used​ ​to​ ​identify​ ​certain​ ​functional groups​ ​within​ ​a​ ​molecule.​ ​Only molecules​ ​that​ ​have​ ​a​ ​dipole moment​ ​will​ ​show​ ​absorbance.​ ​The x-axis​ ​is​ ​reported​ ​in​ ​wavenumbers (reciprocal​ ​centimeters)​ ​and​ ​the y-axis​ ​is​ ​reported​ ​in​ ​percent absorbance. Important​ ​regions: - 1700​ ​-​ ​1750​ ​→​ ​Carbonyls (sharp​ ​peak) - 1720​ ​-​ ​1740​ ​→​ ​Aldehydes - 1700​ ​-​ ​1725​ ​→​ ​Ketones - 1735​ ​-​ ​1750​ ​→​ ​Esters - 1700​ ​-​ ​1725​ ​→​ ​Carboxylic​ ​acids - 3200​ ​-​ ​3600​ ​→​ ​OH​ ​groups​ ​(broad​ ​peak) - 3300​ ​-​ ​3400​ ​→​ ​Amines - The​ ​number​ ​of​ ​peaks​ ​are​ ​relative​ ​to​ ​the​ ​number​ ​of​ ​hydrogens​ ​on​ ​the​ ​amine​ ​(e.g.,​ ​1o​ amines​ ​will​ ​have​ ​two​ ​peaks,​ ​2o​​ ​ ​amines​ ​will​ ​have​ ​one​ ​peak) UV-Vis​ ​Spectroscopy As​ ​the​ ​number​ ​of​ ​conjugated​ ​pi​ ​bonds​ ​increase,​ ​the energy​ ​gap​ ​between​ ​the​ ​highest​ ​occupied​ ​molecular orbital​ ​(HOMO)​ ​and​ ​lowest​ ​unoccupied​ ​molecular​ ​orbital (LUMO)​ ​decreases,​ ​which​ ​means​ ​that​ ​light​ ​of​ ​a​ ​lower energy​ ​is​ ​absorbed.​ ​This​ ​results​ ​in​ ​light​ ​with​ ​a​ ​longer wavelength​ ​to​ ​be​ ​emitted​ ​(as​ ​seen​ ​on​ ​the​ ​right).​ ​If​ ​a molecule​ ​absorbs​ ​green​ ​light,​ ​we​ ​see​ ​it​ ​as​ ​red. Autoradiography Purpose:​ ​To​ ​visualize​ ​the​ ​location​ ​of​ ​a​ ​radioactive​ ​substance​ ​in​ ​a​ ​molecule​ ​or​ ​structure In​ ​the​ ​scope​ ​of​ ​the​ ​MCAT,​ ​autoradiography​ ​is​ ​used​ ​primarily​ ​as​ ​an​ ​imaging​ ​technique​ ​to​ ​identify radiolabeled​ ​atoms​ ​that​ ​are​ ​in​ ​a​ ​molecule,​ ​often​ ​as​ ​part​ ​of​ ​a​ ​Southern,​ ​Northern,​ ​or​ ​Western​ ​blot.​ ​The radiolabeled​ ​substance​ ​is​ ​placed​ ​in​ ​contact​ ​with​ ​a​ ​photographic​ ​emulsion​ ​containing​ ​silver​ ​halide crystals.​ ​The​ ​radiation​ ​of​ ​the​ ​radiolabeled​ ​substance​ ​converts​ ​the​ ​silver​ ​halide​ ​crystals​ ​into​ ​metallic silver,​ ​producing​ ​an​ ​image. X-Ray​ ​Crystallography Purpose:​​ ​To​ ​visualize​ ​the​ ​structure​ ​of​ ​a​ ​molecule,​ ​used​ ​often​ ​with​ ​proteins In​ ​x-ray​ ​crystallography,​ ​a​ ​crystalline​ ​molecule​ ​causes​ ​a​ ​beam​ ​of​ ​incident​ ​x-rays​ ​to​ ​diffract​ ​into​ ​many specific​ ​directions.​ ​A​ ​three-dimensional​ ​image​ ​can​ ​be​ ​constructed​ ​by​ ​measuring​ ​the​ ​different​ ​angles​ ​and intensities​ ​of​ ​the​ ​diffracted​ ​rays. Immunoprecipitation Purpose:​​ ​Used​ ​to​ ​purify​ ​proteins​ ​from​ ​a​ ​solution In​ ​immunoprecipitation,​ ​a​ ​protein​ ​of​ ​interest​ ​can​ ​be​ ​precipitated​ ​from​ ​a​ ​solution​ ​by​ ​adding bead-conjugated​ ​antibody​ ​that​ ​is​ ​specific​ ​to​ ​the​ ​protein​ ​of​ ​interest.​ ​The​ ​antibody​ ​is​ ​bound​ ​to​ ​some​ ​sort of​ ​solid​ ​bead,​ ​which​ ​can​ ​be​ ​used​ ​for​ ​either​ ​extraction​ ​with​ ​a​ ​magnetic​ ​or​ ​by​ ​centrifuge. Radioimmunoassay Purpose​:​ ​Used​ ​to​ ​determine​ ​the​ ​concentration​ ​of​ ​a​ ​protein​ ​of​ ​interest​ ​in​ ​a​ ​given​ ​sample Similar​ ​to​ ​ELISA,​ ​the​ ​wells​ ​of​ ​a​ ​plate​ ​are​ ​coated​ ​with​ ​the​ ​primary​ ​antibody​ ​that​ ​is​ ​specific​ ​to​ ​our​ ​protein of​ ​interest.​ ​Next,​ ​the​ ​radiolabeled​ ​protein,​ ​typically​ ​containing​ ​a​ ​tyrosine​ ​residue​ ​labeled​ ​with​ 125​ ​ I,​ ​is added​ ​to​ ​the​ ​wells​ ​and​ ​binds​ ​to​ ​the​ ​primary​ ​antibody.​ ​The​ ​concentration​ ​of​ ​that​ ​radiolabeled​ ​protein​ ​is determined​ ​by​ ​counting​ ​the​ ​gamma​ ​emission.​ ​Next,​ ​a​ ​sample​ ​of​ ​the​ ​protein​ ​of​ ​interest​ ​in​ ​an​ ​unknown concentration​ ​is​ ​added​ ​to​ ​the​ ​wells,​ ​where​ ​it​ ​competes​ ​for​ ​the​ ​active​ ​site​ ​on​ ​the​ ​primary​ ​antibody, displacing​ ​an​ ​amount​ ​of​ ​the​ ​radiolabeled​ ​protein.​ ​The​ ​concentration​ ​of​ ​the​ ​radiolabeled​ ​protein​ ​is determined​ ​again​ ​by​ ​counting​ ​the​ ​gamma​ ​emission.​ ​The​ ​difference​ ​in​ ​concentrations​ ​provides​ ​the concentration​ ​of​ ​the​ ​protein​ ​of​ ​interest. Mass​ ​Spectrometry Purpose:​ ​Used​ ​to​ ​determine​ ​the​ ​molecular​ ​weight​ ​of​ ​a​ ​compound​ ​and​ ​aid​ ​in​ ​determining​ ​the​ ​molecular structure In​ ​mass​ ​spectrometry,​ ​the​ ​sample​ ​is vaporized​ ​and​ ​subjected​ ​to​ ​ionizing conditions.​ ​The​ ​charged​ ​molecule collides​ ​with​ ​an​ ​electron,​ ​resulting​ ​in the​ ​ejection​ ​of​ ​an​ ​electron​ ​from​ ​the molecule,​ ​making​ ​it​ ​a​ ​radical.​ ​The charged​ ​radical​ ​can​ ​undergo fragmentation​ ​or​ ​being​ ​detected.​ ​The x-axis​ ​represents​ ​the​ ​mass/charge ratio​ ​(m/z),​ ​which​ ​essentially​ ​just means​ ​the​ ​molecules​ ​mass​ ​using​ ​only the​ ​lowest​ ​isotopes​ ​of​ ​the​ ​atoms involved​ ​(e.g.,​ 12​ ​ C,​ 1​​ H,​ 35​ ​ Cl).​ ​The​ ​y-axis represents​ ​the​ ​intensity,​ ​or​ ​relative​ ​abundance​ ​of​ ​the​ ​molecule,​ ​usually​ ​given​ ​as​ ​a​ ​percentage. Base​ ​peak:​​ ​The​ ​tallest​ ​peak.​ ​This​ ​does​ ​not​ ​always​ ​represent​ ​the​ ​actual​ ​intact​ ​molecule,​ ​as​ ​it​ ​may sometimes​ ​be​ ​a​ ​fragment​ ​of​ ​the​ ​molecule​ ​that​ ​is​ ​found​ ​in​ ​higher​ ​abundance. Molecular​ ​ion​ ​peak​ ​(M):​​ ​The​ ​peak​ ​that​ ​represents​ ​the​ ​molecule.​ ​The​ ​m/z​ ​value​ ​of​ ​this​ ​peak​ ​represents the​ ​molecular​ ​weight​ ​of​ ​the​ ​molecule. M+1​ ​peak:​​ ​The​ ​relative​ ​abundance​ ​of​ 13​ ​ C​ ​in​ ​the​ ​molecule.​ ​Found​ ​in​ ​a​ ​relative​ ​abundance​ ​of​ ​1.1%.​ ​So​ ​if there​ ​is​ ​a​ ​M+1​ ​with​ ​a​ ​m/z​ ​value​ ​of​ ​4.4,​ ​it​ ​means​ ​that​ ​there​ ​are​ ​4​ ​carbons​ ​present​ ​(4.4/1.1​ ​=​ ​4). M+2​ ​peak:​​ ​The​ ​relative​ ​abundance​ ​of​ ​either​ 37​ ​ Cl​ ​or​ 81​ ​ Br​ ​in​ ​the​ ​molecule.​ 37​ ​ Cl​ ​will​ ​be​ ​found​ ​in​ ​a​ ​3:1​ ​ratio relative​ ​to​ ​the​ ​M​ ​peak​ ​(e.g.,​ ​if​ ​the​ ​M​ ​peak​ ​has​ ​a​ ​relative​ ​abundance​ ​of​ ​90%,​ ​if​ ​the​ ​M+2​ ​peak​ ​is​ ​at​ ​30%,​ ​it means​ ​there​ ​is​ ​chlorine​ ​present​ ​in​ ​the​ ​molecule).​ 81​ ​ Br​ ​will​ ​be​ ​found​ ​in​ ​a​ ​1:1​ ​ratio​ ​relative​ ​to​ ​the​ ​M​ ​peak (e.g.,​ ​if​ ​the​ ​M​ ​peak​ ​has​ ​a​ ​relative​ ​abundance​ ​of​ ​90%,​ ​if​ ​the​ ​M+2​ ​peak​ ​is​ ​also​ ​at​ ​90%,​ ​it​ ​means​ ​there​ ​is bromine​ ​present​ ​in​ ​the​ ​molecule). Fragments​:​ ​There​ ​are​ ​three​ ​types​ ​of​ ​fragmentation​ ​that​ ​can​ ​occur,​ ​which​ ​can​ ​help​ ​identify​ ​the​ ​structure of​ ​the​ ​molecule.​ ​1)​ ​Alkane​ ​fragmentation,​ ​2)​ ​alcohol​ ​dehydration,​ ​and​ ​3)​ ​alpha​ ​cleavage.​ ​These​ ​are​ ​most likely​ ​beyond​ ​the​ ​scope​ ​of​ ​the​ ​MCAT. Enzyme-Linked​ ​Immunosorbent​ ​Assay​ ​(ELISA) Purpose:​​ ​Used​ ​to​ ​identify​ ​the​ ​concentration​ ​of​ ​a​ ​molecule​ ​of​ ​interest​ ​in​ ​a​ ​given​ ​sample ELISA​ ​is​ ​an​ ​assay​ ​that​ ​uses​ ​primary antibodies​ ​that​ ​are​ ​specific​ ​to​ ​a​ ​molecule​ ​of interest​ ​and​ ​secondary​ ​antibodies​ ​that​ ​are specific​ ​to​ ​primary​ ​antibodies​ ​and​ ​are conjugated​ ​with​ ​a​ ​fluorophore,​ ​so​ ​their presence​ ​can​ ​be​ ​measured​ ​via spectrophotometry.​ ​There​ ​are​ ​two​ ​different methods​ ​of​ ​ELISAs​ ​that​ ​are​ ​used. Indirect​ ​ELISA The​ ​molecule​ ​of​ ​interest​ ​(in​ ​this​ ​case,​ ​considered​ ​an​ ​antigen)​ ​is​ ​coated​ ​to​ ​the​ ​wells​ ​of​ ​a​ ​plate.​ ​A​ ​primary antibody​ ​that​ ​is​ ​specific​ ​for​ ​that​ ​antigen​ ​is​ ​added​ ​and​ ​washed​ ​to​ ​remove​ ​any​ ​unbound​ ​antibodies.​ ​Next, the​ ​secondary​ ​antibody​ ​is​ ​added.​ ​The​ ​secondary​ ​antibody​ ​is​ ​linked​ ​to​ ​an​ ​enzyme,​ ​often​ ​horseradish peroxidase​ ​(HRP),​ ​that​ ​will​ ​be​ ​converted​ ​into​ ​a​ ​fluorophore​ ​when​ ​reacted​ ​with​ ​an​ ​oxidizing​ ​agent,​ ​such as​ ​hydrogen​ ​peroxide.​ ​This​ ​will​ ​cause​ ​a​ ​change​ ​in​ ​color,​ ​which​ ​can​ ​be​ ​detected​ ​using photospectrometry.​ ​The​ ​absorbance​ ​of​ ​the​ ​well​ ​is​ ​compared​ ​against​ ​a​ ​serial-diluted​ ​standard​ ​of​ ​known concentration,​ ​and​ ​based​ ​on​ ​a​ ​determined​ ​curve​ ​using​ ​the​ ​standards,​ ​the​ ​concentration​ ​of​ ​the​ ​molecule in​ ​the​ ​well​ ​can​ ​be​ ​determined. Sandwich​ ​ELISA The​ ​primary​ ​antibody​ ​that​ ​is​ ​specific​ ​to​ ​our​ ​molecule​ ​of​ ​interest​ ​is​ ​coated​ ​to​ ​the​ ​wells​ ​of​ ​a​ ​plate.​ ​Next, the​ ​molecule​ ​of​ ​interest​ ​is​ ​added​ ​to​ ​the​ ​wells​ ​and​ ​then​ ​washed​ ​to​ ​remove​ ​any​ ​unbound​ ​molecules. Next,​ ​the​ ​secondary​ ​antibody​ ​is​ ​added.​ ​The​ ​secondary​ ​antibody​ ​is​ ​linked​ ​to​ ​an​ ​enzyme,​ ​often horseradish​ ​peroxidase​ ​(HRP),​ ​that​ ​will​ ​be​ ​converted​ ​into​ ​a​ ​fluorophore​ ​when​ ​reacted​ ​with​ ​an​ ​oxidizing agent,​ ​such​ ​as​ ​hydrogen​ ​peroxide.​ ​This​ ​will​ ​cause​ ​a​ ​change​ ​in​ ​color,​ ​which​ ​can​ ​be​ ​detected​ ​using photospectrometry.​ ​The​ ​absorbance​ ​of​ ​the​ ​well​ ​is​ ​compared​ ​against​ ​a​ ​serial-diluted​ ​standard​ ​of​ ​known concentration,​ ​and​ ​based​ ​on​ ​a​ ​determined​ ​curve​ ​using​ ​the​ ​standards,​ ​the​ ​concentration​ ​of​ ​the​ ​molecule in​ ​the​ ​well​ ​can​ ​be​ ​determined. Edman​ ​Degradation Purpose:​​ ​Used​ ​to​ ​sequence​ ​the​ ​amino​ ​acid​ ​residues​ ​in​ ​a​ ​protein Edman​ ​Degradation​ ​is​ ​conducted​ ​by​ ​removing​ ​one amino​ ​acid​ ​residue​ ​at​ ​a​ ​time​ ​from​ ​the​ ​N-terminus of​ ​the​ ​peptide.​ ​Phenyl​ ​isothiocyanate​ ​is​ ​added​ ​to the​ ​N-terminus​ ​of​ ​a​ ​polypeptide,​ ​which​ ​then cyclizes​ ​and​ ​breaks​ ​off,​ ​leaving​ ​an​ ​intact polypeptide​ ​that​ ​is​ ​shortened​ ​by​ ​one​ ​residue.​ ​The PTH-amino​ ​acid​ ​hybrid​ ​can​ ​then​ ​be​ ​analyzed​ ​using chromatographic​ ​techniques​ ​to​ ​identify​ ​the​ ​amino acid.​ ​This​ ​can​ ​be​ ​repeated​ ​until​ ​every​ ​amino​ ​acid residue​ ​in​ ​the​ ​polypeptide​ ​is​ ​identified.​ ​This method​ ​is​ ​limited​ ​in​ ​the​ ​fact​ ​that​ ​it​ ​can​ ​only accurately​ ​identify​ ​polypeptides​ ​less​ ​than​ ​50 residues. Gram​ ​Staining Purpose:​ ​Used​ ​to​ ​differentiate​ ​bacteria​ ​into​ ​two​ ​groups,​ ​gram-positive​ ​or​ ​gram-negative,​ ​based​ ​on​ ​the content​ ​of​ ​their​ ​cell​ ​wall The​ ​sample​ ​of​ ​bacteria​ ​is​ ​heat-fixed​ ​to​ ​a​ ​slide​ ​and​ ​crystal​ ​violet​ ​dye​ ​is​ ​added.​ ​Iodide​ ​is​ ​added​ ​to​ ​the bacteria,​ ​which​ ​binds​ ​to​ ​the​ ​crystal​ ​violet​ ​dye​ ​and​ ​traps​ ​it​ ​in​ ​the​ ​bacterial​ ​cell.​ ​The​ ​bacteria​ ​is​ ​then washed​ ​with​ ​alcohol​ ​to​ ​remove​ ​any​ ​dye​ ​that​ ​was​ ​not​ ​taken​ ​up​ ​by​ ​the​ ​bacteria.​ ​Safranin​ ​is​ ​added​ ​as​ ​a counterstain,​ ​so​ ​that​ ​the​ ​bacteria​ ​that​ ​did​ ​not​ ​stain​ ​with​ ​crystal​ ​violet​ ​can​ ​be​ ​visualized. Gram-positive​ ​bacteria:​​ ​Appears​ ​purple​ ​on​ ​the​ ​slide.​ ​Contains​ ​a​ ​thick​ ​peptidoglycan​ ​layer​ ​that​ ​takes​ ​up the​ ​stain. Gram-negative​ ​bacteria:​​ ​Appears​ ​pink​ ​on​ ​the​ ​slide.​ ​Contains​ ​a​ ​thin​ ​peptidoglycan​ ​layer​ ​sandwiched between​ ​two​ ​lipid​ ​bilayers​ ​that​ ​take​ ​up​ ​the​ ​safranin​ ​stain. Restriction​ ​Fragment​ ​Length​ ​Polymorphism​ ​(RFLP) Purpose:​​ ​Used​ ​to​ ​identify​ ​differences​ ​in​ ​homologous​ ​DNA​ ​sequences​ ​based​ ​on the​ ​length​ ​of​ ​fragments​ ​caused​ ​by​ ​restriction​ ​enzymes RFLP​ ​allows​ ​us​ ​to​ ​determine​ ​differences​ ​in​ ​two​ ​or​ ​more​ ​DNA​ ​sequences​ ​of​ ​the same​ ​gene.​ ​Most​ ​often,​ ​this​ ​will​ ​be​ ​comparing​ ​a​ ​wild​ ​type​ ​(WT)​ ​gene​ ​with​ ​a mutated​ ​version​ ​of​ ​the​ ​gene.​ ​The​ ​DNA​ ​strand​ ​is​ ​exposed​ ​to​ ​a​ ​restriction​ ​enzyme, also​ ​called​ ​a​ ​restriction​ ​endonuclease.​ ​Restriction​ ​enzymes​ ​recognize​ ​a​ ​specific palindromic​ ​sequence​ ​(the​ ​sequence​ ​is​ ​the​ ​same​ ​in​ ​the​ ​5’​ ​→​ ​3’​ ​direction​ ​in​ ​both strands)​ ​in​ ​the​ ​DNA​ ​and​ ​cleave​ ​both​ ​strands.​ ​Once​ ​the​ ​DNA​ ​sequence​ ​is​ ​cleaved with​ ​the​ ​restriction​ ​enzymes,​ ​it​ ​can​ ​be​ ​ran​ ​through​ ​gel​ ​electrophoresis.​ ​If​ ​a mutation​ ​has​ ​occurred,​ ​there​ ​may​ ​be​ ​a​ ​difference​ ​in​ ​the​ ​number​ ​and​ ​length​ ​of strands​ ​between​ ​the​ ​WT​ ​and​ ​mutant​ ​genes,​ ​as​ ​the​ ​restriction​ ​points​ ​may​ ​differ. Salting​ ​Out​ ​and​ ​Dialysis Purpose:​​ ​Purification​ ​of​ ​proteins​ ​in​ ​a​ ​solution Adding​ ​salt​ ​to​ ​a​ ​solution​ ​containing​ ​proteins can​ ​allow​ ​you​ ​to​ ​selectively​ ​precipitate​ ​out proteins.​ ​Salting​ ​out​ ​is​ ​due​ ​to​ ​the competition​ ​between​ ​the​ ​salt​ ​ions​ ​and​ ​the protein​ ​for​ ​water​ ​to​ ​keep​ ​the​ ​protein​ ​in solution.​ ​The​ ​salt​ ​concentration​ ​at​ ​which​ ​a protein​ ​will​ ​precipitate​ ​varies​ ​for​ ​each protein. After​ ​a​ ​protein​ ​has​ ​been​ ​precipitated,​ ​the salt​ ​can​ ​be​ ​removed​ ​via​ ​dialysis.​ ​The solution​ ​is​ ​placed​ ​in​ ​a​ ​dialysis​ ​bag​ ​and​ ​then added​ ​to​ ​a​ ​hypotonic​ ​solution.​ ​The​ ​ions​ ​will pass​ ​through​ ​the​ ​semipermeable​ ​membrane of​ ​the​ ​dialysis​ ​bag,​ ​while​ ​the​ ​protein​ ​of interest​ ​is​ ​left​ ​inside. Reducing​ ​Sugar​ ​Tests Purpose:​​ ​Used​ ​to​ ​identify​ ​the​ ​presence​ ​of​ ​a​ ​reducing​ ​sugar​ ​in​ ​a​ ​solution A​ ​reducing​ ​sugar​ ​is​ ​any​ ​sugar​ ​that​ ​is​ ​capable​ ​of​ ​acting​ ​as​ ​a​ ​reducing agent​ ​due​ ​to​ ​the​ ​presence​ ​of​ ​a​ ​free​ ​aldehyde​ ​or​ ​ketone​ ​functional group.​ ​All​ ​monosaccharides​ ​are​ ​reducing​ ​sugars,​ ​since​ ​they​ ​are capable​ ​of​ ​mutarotation,​ ​and​ ​therefore,​ ​will​ ​be​ ​found​ ​in​ ​the open-chain​ ​form​ ​to​ ​some​ ​degree.​ ​However,​ ​not​ ​all​ ​disaccharides, oligosaccharides,​ ​or​ ​polysaccharides​ ​are​ ​reducing​ ​sugars,​ ​since​ ​some glycosidic​ ​bonds​ ​are​ ​between​ ​two​ ​anomeric​ ​carbons​ ​(classified​ ​as​ ​a 1→2​ ​bond),​ ​preventing​ ​mutarotation.​ ​Sucrose​ ​is​ ​an​ ​example​ ​of​ ​a non-reducing​ ​disaccharide​ ​while​ ​maltose​ ​is​ ​an​ ​example​ ​of​ ​a reducing​ ​disaccharide​ ​(as​ ​seen​ ​in​ ​the​ ​figure​ ​on​ ​the​ ​right).​ ​Certain reagents​ ​can​ ​test​ ​for​ ​the​ ​presence​ ​of​ ​a​ ​free​ ​aldehyde​ ​or​ ​ketone group​ ​in​ ​order​ ​to​ ​identify​ ​the​ ​presence​ ​of​ ​a​ ​reducing​ ​sugar. Tollen’s​ ​Test Tollen’s​ ​reagent​ ​tests​ ​for​ ​the​ ​presence​ ​of​ ​an​ ​aldehyde​ ​and​ ​can​ ​distinguish​ ​between​ ​aldoses​ ​and​ ​ketoses. Ketones​ ​do​ ​not​ ​react​ ​unless​ ​they​ ​are​ ​α-hydroxy-ketones.​ ​A​ ​positive​ ​Tollen’s​ ​test​ ​is​ ​characterized​ ​by​ ​the precipitation​ ​of​ ​elemental​ ​silver. Tollen’s​ ​reagent​ ​consists​ ​of​ ​[Ag(NH​3​)​2​]NO​3​. Benedict’s​ ​Test Benedict’s​ ​reagent​ ​tests​ ​for​ ​the​ ​presence​ ​of​ ​an​ ​aldehyde.​ ​Ketones​ ​do​ ​not​ ​react​ ​unless​ ​they​ ​are α-hydroxy-ketones.​ ​A​ ​positive​ ​Benedict’s​ ​test​ ​is​ ​characterized​ ​by​ ​a​ ​change​ ​in​ ​color​ ​from​ ​clear​ ​blue​ ​to brick-red​ ​with​ ​the​ ​formation​ ​of​ ​a​ ​precipitate. Benedict’s​ ​reagent​ ​consists​ ​of​ ​a​ ​mixture​ ​of​ ​sodium​ ​carbonate,​ ​sodium​ ​citrate,​ ​and​ ​copper​ ​(II)​ ​sulfate pentahydrate. Fehling’s​ ​Test Fehling’s​ ​solution​ ​tests​ ​for​ ​the​ ​presence​ ​of​ ​an​ ​aldehyde.​ ​Ketones​ ​do​ ​not​ ​react​ ​unless​ ​they​ ​are α-hydroxy-ketones.​ ​A​ ​positive​ ​Fehling’s​ ​test​ ​is​ ​characterized​ ​by​ ​a​ ​change​ ​in​ ​color​ ​from​ ​clear​ ​blue​ ​to brick-red​ ​with​ ​the​ ​formation​ ​of​ ​a​ ​precipitate. Fehling’s​ ​solution​ ​consists​ ​of​ ​two​ ​parts;​ ​Fehling’s​ ​A​ ​and​ ​Fehling’s​ ​B.​ ​Fehling’s​ ​A​ ​consists​ ​of​ ​aqueous copper​ ​(II)​ ​sulfate.​ ​Fehling’s​ ​B​ ​consists​ ​of​ ​potassium​ ​sodium​ ​tartrate​ ​and​ ​sodium​ ​hydroxide. cDNA​ ​Libraries Purpose:​​ ​Used​ ​to​ ​create​ ​and​ ​house​ ​complimentary​ ​DNA​ ​(cDNA)​ ​strands​ ​that​ ​can​ ​be​ ​expressed​ ​in bacterial​ ​vectors In​ ​order​ ​to​ ​synthesize​ ​proteins​ ​for​ ​things​ ​like​ ​medication,​ ​such​ ​as​ ​insulin,​ ​we​ ​must​ ​clone​ ​the​ ​DNA sequence​ ​for​ ​that​ ​protein​ ​in​ ​a​ ​bacterial​ ​vector.​ ​However,​ ​eukaryotes​ ​have​ ​the​ ​ability​ ​to​ ​undergo post-transcriptional​ ​modification​ ​of​ ​hnRNA​ ​to​ ​create​ ​mRNA​ ​while​ ​prokaryotes​ ​do​ ​not.​ ​Therefore,​ ​we​ ​can not​ ​insert​ ​eukaryotic​ ​DNA​ ​directly​ ​into​ ​a​ ​bacterial​ ​vector​ ​since​ ​it​ ​still​ ​contains​ ​introns.​ ​Instead,​ ​we​ ​must synthesize​ ​a​ ​strand​ ​of​ ​cDNA​ ​from​ ​the​ ​protein​ ​of​ ​interest’s​ ​respective​ ​mRNA. The​ ​mRNA​ ​transcript​ ​of​ ​the​ ​protein​ ​of​ ​interest​ ​is​ ​isolated​ ​and​ ​a​ ​complementary​ ​DNA​ ​primer​ ​containing many​ ​thymine​ ​repeats​ ​and​ ​a​ ​free​ ​3’-OH​ ​group​ ​is​ ​added,​ ​which​ ​anneals​ ​to​ ​the​ ​poly-A​ ​tail​ ​of​ ​the​ ​mRNA. Next,​ ​the​ ​mRNA​ ​is​ ​exposed​ ​to​ ​reverse​ ​transcriptase​ ​and​ ​an​ ​excess​ ​of​ ​the​ ​four​ ​dNTPs​ ​so​ ​a complementary​ ​cDNA​ ​strand​ ​can​ ​be​ ​synthesized,​ ​creating​ ​a​ ​cDNA-mRNA​ ​hybrid.​ ​The​ ​hybrid​ ​strand​ ​is hydrolyzed​ ​using​ ​an​ ​alkaline​ ​solution.​ ​A​ ​primer​ ​is​ ​added​ ​to​ ​the​ ​new​ ​single-stranded​ ​cDNA​ ​and​ ​the complementary​ ​strand​ ​is​ ​synthesized​ ​by​ ​DNA​ ​polymerase.​ ​The​ ​new​ ​double-stranded​ ​cDNA​ ​can​ ​be inserted​ ​into​ ​a​ ​bacterial​ ​plasmid​ ​vector​ ​by​ ​using​ ​restriction​ ​enzymes​ ​and​ ​the​ ​bacteria​ ​will​ ​synthesize​ ​the protein​ ​of​ ​interest.