VARIOUS METHODS OF HOMOGENIZATION

Homogenization

Means to prepare uniform dilution of a known quantity of tissue suspended within a known quantity of a suitable diluents and uniformly macerate or crush the tissue in such a manner as to disperse minute fragments of tissue evenly throughout the mixture.

Tissue homogenization is a process used to prepare tissue samples for certain types of studies.

It involves encouraging the cells to lyse, or break apart to release their contents.

Devices designed for tissue homogenization are used in many lab facilities and specialized techniques may be used for certain types of cells, as for example when cells are difficult to break up because of their structure

Homogenization is one of the steps allowing for preparation of any biological material as a sample for the proteomic analysis. The term “homogenization” covers many meanings such as mixing, stirring, dispersing, emulsifying, but in general, it means: receiving sample of the same composition and structure in the whole volume. By applying homogenization in the procedure of sample preparation, we assume that the sample should change its physical properties without any changes in the chemistry of components.

There are certain variables to be considered while designing a homogenizer/cell disruptor. They are:- type of homogenizing valve/orifice- operating pressure- stages of disruption- viscosity of the sample- temperature

Homogenization methods used for the proteomics purposes can be divided into five major categories:

1. Mechanical;2. Ultrasonic;3. Freeze–thaw;4. Osmotic and detergent lysis

1-Mechanical homogenization.

Rotor–stator homogenizers are one of the best homogenizing tools applied in the laboratories.They can homogenize samples in the volumes from 0.01 ml to about 20 l, depending on the tip and power of a motor applied. Homogenizing tips can easily be cleaned and sterilized

Heat transfer to the processed mixture is low to moderate but usually needs external cooling.

Sample loss is minimal in comparison to pressure processors (French presses), and very small amounts of samples can easily be homogenized.

During operation, the suspended material is drawn into the core of the homogenizer by a rotor turning at 30,000 rpm.

The material is repeatedly cycled through six narrow slits in the stator where it is rapidly sheared and disintegrated by high frequency mechanical action.

Complete homogenization of tissues (muscle, liver, breast tissue, etc.) is usually achieved in ten to thirty seconds.

Ultrasonic homogenizers, also called as disintegrators or sonificators, are based on the electric effect while generating the high energy or ultrasonic wave, interacting with the sample. Energy, resolved after explosion/implosion of gas microbubbles, effectively destroys solid particles such as cells.

Ultrasonic devices are mainly used to homogenize small pieces of soft tissues (brain, blood, liver).

Tough and dense tissues are not recommended to homogenize using this equipment.

Ultrasonic homogenizers

This type of homogenization uses effect of ice crystals formation in the tissue during freezing process.

The method is relatively fast, effective and, what is also important, does not introduce any external impurities into the sample because freezed solution is isolated from the external environment.

Freeze–thaw homogenization

These methods of disruption of cells utilize osmotic pressure or detergent interactions to destroy cells’ walls and membranes. They are also efficient for homogenization of nuclear and mitochondrial membranes in cell extracts.

Osmotic lysis is often used to disrupt blood cells. It may be useful for RNA extraction, even from bacteria like Brucella abortus internalized in macrophages

Depending on the conditions, osmotic lysis can be used also for microbial cell disruption.

Osmotic and detergent lysis

 This image shows how the detergent molecules interefere with the lipids and proteins on the cell membrane and nuclear membrane, causing the DNAto be more exposed.