BMB 251 1st Edition Lecture 6Outline of Last Lecture I. Proteinsa. Side chains:i. Acidicii. Basiciii. Uncharged, polariv. NonpolarII. Polypeptide foldinga. Noncovalent bondsIII. Configuration IV. ComplexesV. Four levels of organizationVI. Restrictions of 3D movementsOutline of Current Lecture VII. Purification of a ProteinVIII. HomogenatesIX. Preparative ultracentrifuge X. Column chromatographyXI. High Performance Liquid Chromatography (HPLP)XII. Affinity chromatographyXIII. Fusion proteinXIV. Tandem Affinity Purification-Tapping (Tag-tapping)XV. Sodium dodecyl sulfate (SDS) Gel electrophoresisXVI. Westerner blotting/immunoblottingCurrent Lecture- The purification procedure usually starts with subcellular fractionation to reduce complexity and then is followed by purification steps that increase specificity- To purify a protein, proteins must be extracted from cell and cells have to be broken up o Protein purification: accomplished through serial separation stepso In order to determine a protein’s structure and function, a large quantity of the protein is needed and all of the contaminants must be removed- Homogenate/extract: suspension of cells reduced to think slurryThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Preparative ultracentrifuge: homogenate is separated by rotating extracts of broken cells at high speeds separates cell components by size and density (largest units experience the largest forces and are deposited at the bottom of the tube)o Highest speeds can create a “pellet” (leftover remains in test tube to be examined) containing ribosomes, viruses, large macromolecules, proteins; this is very expensive- Column chromatography (way in which proteins are most often fractionated): mixture of proteins in solution is passed through a column containing porous solid matrix retarded to different extents collected separately as they flow out the bottom of the column- High Performance Liquid Chromatography (HPLP): attain high degree of resolution; method of choice for separating many proteins and small molecules- Affinity chromatography: takes advantage of building interactions occurring on protein surface- Fusion protein: an enzyme might be bound to a protein substrate molecule binds tightly to enzyme fusion protein is isolated in cell- Tandem Affinity Purification-Tapping (Tag-tapping): one end of the protein contains two recognition tags separated by a protease cleavage site- Sodium dodecyl sulfate (SDS) Gel electrophoresis: very common method for separating proteins (or nucleic acids)o Electrical charge applied to solution containing a protein molecule causes protein to migrate at a rate depending on its net charge, size and shapeo Powerfully negatively charged detergent (SDS) binds to hydrophobic regions of proteins, causing them to unfold into polypeptides chains and individual protein molecules are freed from other protein, lipids, etc.o Protein binds with negatively charged detergent moves along gel to the more positiveside; smaller molecules move farther down gel while larger molecules get stucko All proteins are easily detectable when treated with blue dye (aka Coomassie Brilliant Blue) making them very visibleo SDS separates ALL types of proteins, even ones that are normally insoluble in water (such as ones that are in the cell membrane)- Westerner blotting/immunoblotting: method of detecting proteins by exposing proteins to antibodies coupled with radioactive isotope to easily detectable enzyme o The primary antibody detects while the secondary antibody amplifies and
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