Describe the technique you would use to separate these proteins. Be specific and

Question

Describe the technique you would use to separate these proteins. Be specific and note what should be on the solid matrix of the column (+ or - charge, hydrophobic bit, ATP, whatever) and what solution you would use to elute the proteins that stuck to the column off the column. Warm up: How would you separate protein Falcon-ase which is large and positively charged from protein Braves-ase which is small and neutral charge? If you have a mixture of protein Braves-ase, Falcon-ase (from problem 1) and Hawkish which is the approximately the same size as Braves-ase but positively charged, how would you purify each from mixture? Larry Labtech grew cells, broke them open with a blender in a buffer at pH 7, and removed the cell debris by centrifugation. Larry then added (NH_4)_2 SO_4, to the protein solution to 40% saturation (about 1.6 M (NH_4)_2 SO_4), and centrifuged out a precipitate. To the remaining supernatant, he added (NH_4)_2 SO_4 to 70% saturation, and spun down the precipitate. Each of the two pellets was redissolved in a buffered solution. Larry thus has three samples: the supernatant, pellet #1 and pellet #2. Describe the principle of (NH_4)_2 SO_4 fractionation. Would you expect all proteins to precipitate at the same concentration of (NH_4)_2 SO_4? Why or why not? The next purification step involved gel filtration to separate the proteins from the supernatant fraction. In gel filtration, the elution volume of a sample is proportional the molecular weight of protein. Larry Labtech evaluates the gel filtration column with calibration standards to determine relative volumes, then samples are applied to the column. Skipping some arithmetic to arrive at a rough estimate, Larry developed the following data table.

Explanation / Answer

Answer 1. The technique to be used is GEL ELECTROPHORESIS. This one is useful when we have different proteins based on charge and size to separate. In this method, mixture of proteins is subjected to a gel formation and an electric current is applied to the system. This will, in this case, led to the separation of smaller, neutral protein as it will migrate faster in the gel column. But the larger, positively charged protein will not be able to move through and thus, can be separated. Generally, column gel is like a matrix, composed of polyacrylamide. The elution methods can be via diffusion or electroelution.

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