A sonicator probe is lowered into the suspension of cultured cells and high frequency sound waves
(> 20 kHz) generated for a short duration that causes disruption of cells by shear force and
cavitation. Cavitation are areas undergoing alternate compression and rarefaction, that rapidly
interchange. The gas bubbles in the buffer are initially under pressure but, as they decompress,
shock waves are released which disrupt the cells. Only small samples (50–100 cc) can be handled.
As heat is produced during sonication, it becomes imperative that samples be kept on ice during
treatment.
(NH
4
)
2
SO
4
precipitation: Upon the addition of salt (<0.15 M) the solubility of proteins increases,
an effect termed salting-in. However, at higher salt concentrations, protein solubility decreases and
thus they precipitate; this effect is termed salting-out.
2.2. Hydrophobic interaction chromatography
Source taken from: Principles and techniques of Biochemistry and Molecular Biology (7th
edition) by Keith Wilson and John Walker.
This type of chromatography exploits the surface hydrophobicity of proteins. In pure water, the
water molecules cover the hydrophilic regions of the proteins thus masking the hydrophobic
groups located in the core of the protein. However, upon addition of salt, water is used to solvate
the salt molecules as a consequence of which hydrophobic groups on the proteins are exposed
which can interact with hydrophobic groups (alkyl or aryl) coupled to the matrix. Commercially
available matrices include Phenyl Sepharose, Phenyl SPW etc. A porous matrix is used to provide
high internal surface area which is equilibrated with the buffer having the same salt concentration
as in our sample. The type of salt and its concentration in the start buffer depends on whether the
proteins of interest bind to the column and majority of contaminating proteins pass directly through
the column. Proteins are eluted by decreasing the salt concentration in the elution buffer. As the
level of salt decreases proteins with the lower hydrophobicity are the first to be eluted from the
column. Proteins can thus be eluted differentially in a purified, concentrated form. Proteins with
the highest hydrophobicity will be most strongly retained and will be eluted last. The salt-free
buffer helps remove most tightly bound proteins at the end of an elution.
Gel filtration chromatography: It is also known as size exclusion chromatography and involves
the separation of molecules based on their size. Matrix here consists of porous beads which can be
made of silica, polyvinyl acetate, cross linked dextran etc. Pore size to be used depends on the size