Question

What is the crude cell extract? Name and explain two methods of partially purifying the crude cell extract.?

What is the chemical basis of ion exchange chromatography? What is a cation exchange solid support? What is an anion exchange solid support? Why do some proteins “stick” to a cation exchange solid support, while others stick to an anion exchange solid support? How are proteins that “stick” to the solid support released in ion exchange chromatography?

Answer 1

Q1: The cells are made up of different biomolecules including proteins, lipids, carbohydrates, nucelic acid, metals and ions. If we are intrested to extract and isolate these biomolecules, then a cell/tissue has to be disrupted either physically or chemically inorder to relase them as an extract. Such combination of a mixture containing different cellular components is known as crude cell extract.

The choice of the methods used for purification of cell crude extract depends upon the type of biomolecule a person is intrested with. The fractionation techniques involved work on the principle of solubility, binding affinity, and charge. There various methods used to partially cell crude include chromatography (Ion exchange, column, Dialysis, centrifugation, HPLC etc.

1. Dialysis: The sepeartion of biomolecules like proteins can be done using a semi-permeable membrane which allows smaller molecules to pass (<10kDa) and not the protein molecules.

2. Centrifugation: The separation of molecules occurs based on the molecular mass when subjected to centripetal forces. The various versions of centrifuges like ultra-centrifugation, equilribrum density gradiemt centrifugation, rate-zonal centrifugation etc.