The table shows the data for purification of a hypothetical protein. Determine the missing values for the specific activity, yield, and purification level for the three types of chromatography. | Step | Total protein (mg) | Total activity (units) | Specific activity (units/mg) | Yield (%) | Purification level | | :--- | :--- | :--- | :--- | :--- | :--- | | Homogenization | 15100 | 1.35 × 10^5 | 8.94 | 100 | 1 | | Salt fractionation | 4610 | 1.23 × 10^5 | 26.7 | 91.1 | 2.99 | | Ion-exchange chromatography | 1.20 × 10^3 | 1.01 × 10^5 | | | | | Gel-filtration chromatography | 67.5 | 70300 | | | | | Affinity chromatography | 3.85 | 45800 | | | |
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Specific activity is defined as the total activity (units) divided by the total protein (mg). So, we can calculate the specific activity for Ion-exchange chromatography and Affinity chromatography as follows: Ion-exchange chromatography: Specific activity = Show more…
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The table shows the data for purification of a hypothetical protein. Determine the missing values for the specific activity, yield, and purification level for the three types of chromatography: | Step | Total protein (mg) | Total activity (units) | Specific activity (units/mg) | Yield (%) | Purification level | |------------------------|--------------------|-----------------------|------------------------------|-----------|--------------------| | Homogenization | 15100 | 1.35 x 10^5 | 8.94 | 100 | 1 | | Salt fractionation | 4610 | 1.23 x 10^5 | 26.7 | 91.1 | 2.99 | | Ion-exchange chromatography | 1.20 x 10^3 | 1.01 x 10^5 | | | | | Gel-filtration chromatography | 67.5 | 70300 | | | | | Affinity chromatography | 3.85 | 45800 | | | |
Dennis H.
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 $\mathrm{Mb}$, obtained at a given step ( $\mu \mathrm{mol}$ 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 $(\mu \mathrm{mol}$ 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 step-DEAE or affinity chromatography-causes 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?
Maitreya E.
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