Question 16. What is “salting in”? “Salting out”? Explain the mode of action of

Question

Question 16. What is “salting in”? “Salting out”? Explain the mode of action of ammonium sulfate in the context that you are familiar with thru AP purification.

Question 17. You intend to precipitate various proteins from plasma using ammonium sulfate using the table below and the table at the end of the quiz (nomogram for determining the amount of ammonium sulfate needed to attain respective percentages of saturation.). How much ammonium sulfate powder do you need to add to precipitate each one of three fractions (fibrinogen, globulins, albumins)? Complete the last column in the table below.

Question 19. Explain the procedure of western blotting. Draw a schematics of WB sandwich, indicate poles, and explain what happens if the poles were reversed.

Solubility of plasma proteins in (NH42SO4 solutions Saturation by Protein fraction Salt required (NH4)2SO4 precipitated (g/100 m) 0-20 Fibrinogen Globulins 20-50 50-100 Albumins

Explanation / Answer

"Salting in" refers to the observation , the solubility of the protein increases at dilute solutions of low salt concentrations. As the solubility of the salt added in higher than protein, then salt is more likely dissolve and occupied most of the space in the solution; therefore, proteins aggreate and precipitate.

Salting out is an effect based on the electrolyte-nonelectrolyte interaction, in which the non-electrolyte i. e protiens could be less soluble at high salt concentrations and thus aggreates and precipitated out.

Ammonium sulfate enhances the solubility of the alkaline phosphate (salting in effect) and decreases the solubility of the ammonium phosphate salts (salting out effect). Thus ammonium phosphate precipitates out and alkaline phosphate is extracted from the solution. And we get alkaline phophate in pure form.

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