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- 10. A new protein of unknown structure has been purified. Gel filtration chromatography reveals that the physiological protein has a molecular weight of 240,000 Daltons. Chromatography in the presence of 6 M urea yields a single peak corresponding to a protein of 60,000 Daltons. Chromatography in the presence of 6 M urea and 10 mM mercaptoethanol yields peaks for proteins of 34,000 Daltons and 26,000 Daltons. What can we learn about protein's tertiary and quaternary structure from this data?What is the function of sodium dodecyl sulfate (SDS) in SDS-PAGE? stabilizes the gel matrix, improving resolution during electrophoresis SDS solubilizes proteins to give them uniformly negative charges, so the separation is based purely on size. SDS raises the pH of the gel, separating multiunit proteins into individual subunits. SDS solubilizes proteins to give them uniformly positive charges, so separation is based purely on pH.B W S sult, it makes the protein Why is denaturing the proteins necessary for SDS PAGE to work? Use YOUR OWN X mand 3 E D с $ R F 5 Lê 6 T G MacBook Pro B Y H U N J 8 I M 9 K O I O L 44 P command . : 8 ; x { + C option ? I } 1 M
- SDS-PAGE gels are useful in determining the molecular weights of proteins; however, the molecular weights of are usually not reliable. protei1. In Gel filtration chromatography, when will you stop collecting eluents if sample is not colored? 2. How does SDS-PAGE separate proteins and peptides from each other? Explain. 3. Explain the Donnan Membrane Phenomenon. Why is it important for the homeostasis of the cell?I. A protein, X, was Isolated from a pathogenlc mlcroorganism. The proteln Is a vlrulence factor whose path0genlclty lies In a heptapeptide of unknown sequence. After trypsin cleavage of the heptapeptide from protein X, the peptlde's compOsition and sequence was determined. The fOllowing were the results of the sequenclng process: 1. When the peptide was treated with dinitrofluorobenzene (DNFB), DNP-asp and a mixture of amino acids were produced. 2. When the same Intact peptide was treated with streptococcal protease, a pentapeptide of composition asp, asN, cys, gly and ser and 2 amlno acids were released. 3. When the heptapeptlde was also treated with hydrOxylamine HCI, a tripeptide and a tetrapeptide were obtained. The C-terminal amino acid of the tripeptide was asN. 1) What is the sequence of the heptapeptide if it is composed of cys, asp, lys, asN, gly and ser only? 2) What is the pl of the heptapeptide?
- In size exclusion chromatography, which component will elute last in the set up? O Small proteins will elute last because they do not pass through the pores of the gel beads. O Large proteins will elute last because they pass through the pores of the gel beads. O Small proteins will elute last because they pass through the pores of the gel beads. Large proteins will elute last because they do not pass through the pores of the gel beads.Configurational antrapy Molten globules Clusters of frustrated contacts Functionally siatinet states B Which is the site of folded proteins O A BWhy do we use a stacking gel in SDS-PAGE
- 2 a. You are trying to purify protein C from a mixture of proteins noted in the above Table. If you had only one type of column to choose from, which one would allow you to purify protein C with the least number of contaminants? Size exclusion column Ion exchange column Affinity chromatography using glucose as the bait Affinity chromatography using NAD as the bait Please explain why you chose the column above based upon the properties of the column AND the proteins in the Table.4. Deducing quaternary structure via SDS-PAGE. SDS-PAGE is a convenient method for separating polypeptides solely on the basis of size. Small polypeptides travel faster than large ones; rate of migration through the gel is inversely proportional to the logarithm of molecular weight. The subunit structure of a multimeric protein often can be deduced using this technique in conjunction with a protein cross-linking agent. Cross-linking agents react with amino acid residues of two polypeptides that are in contact, thereby linking them by covalent bonds. After limited treatment with the reagent, so that some but not all subunits become cross-linked to their neighbors, the protein is subjected to SDS-PAGE and the molecular weights of the resulting proteins (bands) are estimated. The results of such experiments on two proteins (Protein A and Protein B) are shown below. What is the most likely subunit structure of each protein? Protein A SDS-PAGE cross-linking agent MW 105,000 70,000 35,000…1. Calculate the molecular weight of protein in sample A. Given the protein migration distance is 26 mm and the migration distance of the dye front (gel length) is 6.5 cm.