BBMB404Chapter3

BBMB404Chapter3 - Chapter 3: Exploring Proteins and...

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Unformatted text preview: Chapter 3: Exploring Proteins and Proteomes Protein function L o t s o f F u n c t i o n s How many proteins are there? A lot. Currently data from the Human Genome project estimates that humans have 20,000-25,000 genes, but due to alternate splicing, and post- translational modifications including cleavage and chemical modification, it is now estimated that there may be as many as 1,000,000 different protein products.in humans alone. A group studying human blood plasma estimates that there are probably some 100,000 plasma proteins, but as of 2004 only about 1000 have been detected and studied. How do we learn the functions of all these proteins? After all, thats what biochemistry is all about. Studying protein structure and function A typical experimental format: 1. Develop an assay to identify and quantify the protein (or its activity) 2. Select a biological source of the protein and prepare the cell homogenate 3. Separate the cell homogenate into fractions by fractional centrifugation 4. Chromatography [ion exchange, gel filtration, affinity] 5. Determine proteins purity and molecular size 6. Carry out further investigation 1. Break open cells 2. Gross fractionation, usually based on size Differential centrifugation 3. Subfractionation based on size, charge (hydrophobicity), and affinity. Protein purification: how do you do it? Protein purification: size exclusion by dialysis Liquid Chromatography Collect fractions Different Proteins migrate at different rates Stationary phase Solid packed in column Migration through solid differs according to properties Ion exchange Gel filtration Affinity Mobile phase Liquid medium that transports sample through column Protein purification: size exclusion column chromatography (or gel-filtration ) Sephadex, Sepharose, Bio-gel: ~ 100M beads of insoluble carbohydrate matrix like agarose, dextran, or polyacrylamide. ein purification: hydrophobic interaction chromatography ( HIC) ed on hydrophobicity At high salt concentrations, water is even more in demand for polar interactions This effectively increases hydrophobic interactions. Ion exchange chromatography based on charge This time the beads have charged groups, either positive or negative. By varying pH and salt, proteins can be separated by exploiting their differing charges and charge densities. Anion exchange pH 7 to ensure charge Cation exchange pH 4.4 to ensure charge At pH 7, which is retained most: KKKKK or DDDDD? At pH 7, which is retained most: KKKKK or DDDDD? Protein charge varies with pH and sequence, and the strength of interaction with the column depends on the ionic strength of the solvent. Affinity chromatography based on unique binding behavior Antibodies, metal-chelation/ tagging (e.g. His-tag) H 2 N CH C CH 2 OH O N N Ni NH 2 CH C H 2 C HO O N N H 2 N CH C H 2 C OH O N N NH 2 CH C CH 2 HO O N N His tags (6xHis at N- or C-terminus) are very common these days HPLC: High Pressure (or Performance) Liquid Chromatography...
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BBMB404Chapter3 - Chapter 3: Exploring Proteins and...

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