Question

please i want computer typing answer for A and B

The following techniques will be used for purification of an enzyme from cultured cells; salt fractionation, affinity chromatography, ion-exchange chromatography, homogenization and gel filtration chromatography. The protocol states that the techniques should be used in the order listed above.

a) What is wrong with the order of techniques as they are listed?

b) How does each technique contribute to the process of obtaining a pure enzyme?

Answer 1

Answer a: The order of techniques is wrong for the purification of an enzyme from cultured cells. First homogenization has to be done. This should be followed by salt fractionation, gel filtration chromatography, ion-exchange chromatography and then affinity chromatography. This order will help in purifying protein from lowest to highest purity.

Answer b: First of all, homogenization should be done. Homogenization disrupts the cell wall to release the enzymes into the homogenates along with other cellular components. In the second step, salt fractionation should be done to precipitate proteins. In the third step, gel filtration chromatography technique should be used in the separation of large and small proteins. Then, ion-exchange chromatography should be used that separates proteins based on their net charges. Affinity chromatography is highly is selective and should be used at the last in the order. Specific ligands are used that used in the column and the target protein is adsorb from the extract as it passes through the column, while the rest is washed away. Affinity chromatography provides the purest enzyme.