Question

1. In the last lab,you prepared the cell lysate for affinity chromatography using three techniques for lysing the cells. Name and briefly describe the three techniques. Also, explain why you didn't use SDS to lyse the cells in this case (as you did for the Western blot.)

Answer 1

Cell lysis can be prepared by 3 techniques, namely
1. Liquid Nitrogen grinding: Protein sample is blended with liquid Nitrogen until they freeze and become brittle. The sample is then ground using a pestle and mortar. Grinding in liquid nitrogen is effective in plant or fungi samples.
2. Bead beaters: Sample is combined with tiny beads in a bowl and mechanically then agitated in order to break the cells apart. The sample should be allowed to rest and cool inbetween the beating cycles, so as to avoid over-heating of the sample.
3. Lysis buffers: A specific lysis buffer can be used to specifically break open cells or tissues chemically. It could be either lytic enzymes, reducing agents, salts or detergents to release proteins or other cellular components. One such example is Tris-HCl.

In Affinity Chromatography technique, one should not use ionic detergents such as SDS (anionic detergent). This is because the SDS tends to bind to the anion-exchange support system of the affinity chromatography, that is the stationary phase (could be agarose gel, etc.)