Chapter 5: Q14CP (page 119)
What types of information can be retrieved from a protein sequence database?
The protein sequence database retrieve protein sequence records by gene name, organism, author name, accession number and a variety of other text terms.
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Explain how an antibody could be useful for purifying a protein and for determining its concentration.
A pentapeptide has the sequence(using one-letter symbols for amino acids). Calculate the mass of this peptide, as determined by mass spectrometry, to three significant figures.
Identify the first residue obtained by Edman degradation of cytochrome c from (a) Drosophila, (b) bakerโs yeast, and (c) wheat germ (see Table 5-6).
Consult Table 5-1 to complete the following: (a) On a plot of absorbance at 280 nm versus elution volume, sketch the results of gel filtration of a mixture containing human cytochrome c and bacteriophage T7 RNA polymerase and identify each peak. (b) Sketch the results of SDS-PAGE of the same protein mixture showing the direction of migration and identifying each band.
Question: Purification tables are often used to keep track of the yield and purification of a protein. The specific activity is a ratio of the amount of the protein of interest, in this case Mb, obtained at a given step (ฮผmol or enzyme units) divided by the amount (mg) of total protein. The yield is the ratio of the amount of the protein of interest obtained at a given step (ฮผmol or enzyme units) divided by the original amount present in the crude extract, often converted to percent yield by multiplying by 100. The fold purification is the ratio of the specific activity of the purified protein to that of the crude preparation.
(a) For the purification table below, calculate the specific activity, % yield, and fold purification for the empty cells.
(b) Which stepโDEAE or affinity chromatographyโcauses the greatest loss of Mb?
(c) Which step causes the greater purification of Mb?
(d) If you wanted to use only one purification step, which technique would you choose?
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