26-03-2014, 02:26 PM
WESTERN BLOTTING
DEFINATION OF WESTERN BLOTTING
The western Blotting) is a analytical technique used to detect specific proteins in the given sample.
Alternately called protein immunoblotting.
The Western blotting method was first described in 1979.
OTHER BLOTTING TECHNIQUES
This family of related techniques has
continually expanded to include :
Eastern blotting :
(Post-translational modifications).
Far Western blotting :
(Protein: protein interactions)
Far Eastern blotting :
(Lipid detection).
STEPS
Western blot sample Preparation
Gel electrophoresis
Transfer of proteins
Antibody probing
Detection
Imaging
Analysis
SAMPLE PREPARATION
Numerous methods are available for disrupting cells and preparing their contents for analysis by Western blotting.
In general gentle methods are employed for easily lysed cultured cells or blood cells.
More vigorous methods are employed for the disruption of:
Bacterial cells
Plant cells or
Mammalian cells
Gel Electrophoresis
Electrophoresis is a separation technique based on the :
Mobility of charged molecules in an electric field.
It is used mainly for the analysis and purification of large molecules such as proteins or nucleic acids
CHARGE ON PROTIEN MOLECULES
Proteins are amphoteric (or zwitter ionic) compounds.
They are therefore either positively or negatively charged because they contain both acidic and basic amino acid residues.
For each type of protein, there is a pH at which the molecule has no net charge ,this is called is isoelectric point
At normal electrophoresis group will tittered and protein will have zero net charge.
Wet transfer
In this choice of transfer the gel and membrane are both fully immersed in transfer buffer .
Then current is applied in the direction of the gel to the membrane.
Generally, wet transfer requires:
Cooling of the unit
Internal recirculation of the transfer buffer by the presence of a stirring magnet.
Wet transfer is recommended for large proteins.
It is a relatively slow technique.
Requiring large volumes of buffer
Quantitative protein analysis
Western blotting has long been used for qualitative protein analysis to confirm protein presence and estimate protein amount.
The development of highly sensitive detection reagents and advanced imaging techniques has made Western blotting a potential tool for quantitative protein analysis.