02-Exploring Proteins

02-Exploring Proteins - Exploring Proteins The proteome is...

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Exploring Proteins The proteome is the functional representation of the genome. Proteome: proteins expressed by the genome. Represents functional expression of information, which varies with cell type, developmental stage, and environmental conditions. Takes into account alternatively spliced RNA, post-translational modifications, temporal regulation of protein synthesis, and varying protein-protein interactions. Protein purification is the indispensable first step. Separation is achieved based on solubility, size, charge, and binding ability. Determine sequence: automated peptide sequencing and/or recombinant DNA methods. Locating the protein in physiological context by antibody binding. Obtaining the 3D atomic resolution structure by NMR (nuclear magnetic resonance) spectroscopy and/or x-ray crystallography. Peptide synthesis enables the synthesis of new drugs, functional protein fragments, or antigens.
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Where is the Protein? Assay: the test to uniquely identify the protein to determine the presence of the protein of interest. For enzymes (protein catalysts), the assay is based on the reaction the enzyme catalyzes in the cell. Lactate dehydrogenase catalyzes the conversion between lactate and pyruvate while reducing NAD + to NADH. NADH (nicotinamide adenine dinucleotide) uniquely absorbs light at 340 nm. The progress of the reaction can be followed by monitoring the absorbance of the reaction mixture at 340 nm. The amount of total protein in the assayed mixture is also determined. Specific activity: the ratio of enzyme activity over amount of protein. The specific activity will rise as the purification proceeds. The point of purification is to maximize specific activity, thereby obtaining a pure sample of the desired protein.
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Releasing Proteins from Cells Disruption of cell membrane to form the homogenate. Differential centrifugation The mixture is fractionated by centrifugation to give a dense pellet of material at the bottom and the supernatant above. The supernatant is centrifuged again with greater force to give another pellet and supernatant. Fractions of decreasing density are obtained with one fraction enriched with the desired protein/activity.
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Salting Out and Dialysis Salting out: most proteins are less soluble at high salt concentrations. The concentration at which a protein precipitates differs from protein to protein. Fibrinogen: 0.8 M NH 4 SO 4 . Serum albumin: 2.4 M NH 4 SO 4 . Hofmeister series: salting out effectiveness Na 3 (citrate)>Na 2 SO 4 ~K 2 HPO 4 >(NH 4 ) 2 SO 4 >Na(acetate)>NaCl>NaNO 3 Dialysis can be used to remove the salt. Dialysis: use of semipermeable membrane to separate proteins from small molecules. Molecular larger than the pores are retained inside the dialysis bag.
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02-Exploring Proteins - Exploring Proteins The proteome is...

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