aii Disadvantages aii1 Protein Concentration can be greatly underestimatedOver

Aii disadvantages aii1 protein concentration can be

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a.ii)Disadvantages(a.ii.1)Protein Concentration can be greatly underestimated/Over estimated(a.ii.1.a)Sample contains proteins that are either rich in tyrosine, tryptophan, and phenylalanine amino acids, or areparticularly poor can cause inaccurate results(a.ii.2)Many Non-Protein impurities(a.ii.2.a)Can cause absorptionb)Biuret Method’b.i)Cu2+ ions interact with peptide bonds under appropriate conditions to
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give a blue color, which can be detected spectrophotometrically b.ii)Disadvantages(b.ii.1)Ammonia and Tris-Buffers interfere with this method. Thus this method can not be used to determine protein concentration ofproteins that have been obtained by ammonium sulfate precipitation(b.ii.2)Does not detect low concentrations of proteinsc)Lowry Methodc.i)Phosphotungstate & Phosphomolybdateions are added to the CU2+-protein (Biuret) Complex, which results in blue colorc.ii)The color comes from two structural features(c.ii.1)Copper Complexedwith peptide bonds(c.ii.2)Molybdate Tungstate Complexesthat are produced when these compounds interact with tryptophan, tyrosine, and polar amino acids(c.ii.3)Method is highly accurate and detects low concentrations of proteins(c.ii.4)Disadvantages:(c.ii.4.a)Standard curve must be prepared(c.ii.4.b)preparation requires many steps and is more time consuming(c.ii.4.c)variety of molecules commonly present in cell extracts or used as buffers can interfere with absorption d)Bradford Assayd.i)Brown dye, Bradford Reagent,that binds specifically to proteins; after binding to proteins - turns blue, 595nm abs.d.ii)Method is accurate and detects low concentrations of proteins6)Purifying Proteins from crude Extracta)When cells are broken up and proteins are released, many other kinds of molecules are also released in the samples solutionb)Purification requires a series of techniques, each of which removes some types of unwanted molecules and retains the molecules of my interestc)Ammonium Sulfate Precipitationc.i)High concentration of salt, cause most proteins to precipitatec.ii)Salt is added in step wise fashion to point of saturation(c.ii.1)each additional increment of salt, certain classes of crude extract proteins precipitate out of the solution (c.ii.1.a)precipitates are removed by centrifuge and resuspended in a buffer solution7)Electrophoresis Lab Questions
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Protein Extraction Buffer ContainsPIPES BufferMaintains constant pHEGTAChelator of Ca2+/ Slows down protein degrading processMgCl2Ionic BalanceDTTReducing Agent, prevents oxidative damage of the proteins LeupeptinProteases Inhibitor (Inhibits Enzymes that chop up proteins)PepstatinProteases Inhibitor (Inhibits Enzymes that chop up proteins)NaN3Sodium Azide, Preservative Biocide, prevents bacterial growth, TOXIC 4X Gel Loading Buffer ContainsBromophenol Blue“Tracking Dye” Marks migration front on an electrophoresis gelSDS
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