assays in Biochem

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assays in Biochem
2011-09-26 09:22:09
assays separation techniques

separation techniques of proteins.
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  1. Discribe what a Lowry assay is.
    The Lowry assay utilizes copper in solution and binds to peptide bonds. 3 aminoacids must be present to work. The more color is in solution the more peptide bonds you have. Make a calibration curve from standards to discover unknown.
  2. What is an enzyme assay?
    track the activity of an enzyme by measuring the product that is formed.
  3. Discribe how mass spectrometry can be used to determine molecular mass.
    The proteins are ionized and vaporized and are injected into a matrix of MALDI-TOF. Standards have been timed on how long it takes them to travel through the matrix.
  4. discirbe edmand degragation
    uses phenylisothiocanate (PITC). Binds to the N terminaus and sends the amino acid down a column to be identified. amino acids are still intact and come off one at a time
  5. What is FNDB?
    FNDB is 2,4-dinitrophenyl derivative. It lables the N-terminaus but is followed with 6 M HCl which yeilds free amino acids as the rest of the chain. Useful because it lables the N-terminus
  6. How do you find locations of disulfide bonds in amino acids?
    1) Use SDS-PAGE, allow the proteins to run and then turn the gel 90 degrees and watch them spread. Do it again with the same protein, but this time don't use mercaptoethanol so the disulfide bonds will still be intact. When this gel is turned 90 degrees some fragments will not spread, indicating a disulfide bond.
  7. What amino acids from disulfide bonds?