![]() ![]() ![]() Dot blot technique aids in direct blotting of biomolecule onto the membrane.It does not involve immobilization of the biomolecules from a gel matrix to the filter membrane.One can detect the presence or absence of genes from the sample of transgenic individuals in a single test run.Dot blot technique does not require the separation of bands on the solid support medium (agarose), or there is no requirement of electrophoresis.Colourimetric detection: Measure the intensity of colour by using the colourimeter.The addition of substrate will give a specific colour to the sample. Add substrate after the attachment of the secondary antibody with the primary antibody. The secondary antibodies attach with the enzymes. Addition of secondary antibody: The secondary antibody specifically binds with the primary antibodies.Washing: After binding the primary antibody with the target protein, wash the filter paper to wash off the unbound primary antibodies by using the PBS buffer.Addition of primary antibody: The primary antibodies fix with the target protein molecule.Blocking: Then, add bovine serum albumin (BSA) medium or dry milk to block the extracellular space in the filter membrane.Blotting: It involves the addition of different protein sample directly onto the nitrocellulose or PVDF filter membrane.Extraction of Protein: Take out different protein samples from different tissues or cells.The identification of Protein by dot blot technique involves the following steps: Autoradiography: Expose the filter membrane to the X-ray film to visualize the target RNA.Washing: After hybridization, wash the unbound or unhybridized radioactive probe from the filter medium.The radioactive probe will complementarily pair with the target RNA or hybridize the RNA. Hybridization: Add the radioactive probe to the filter medium containing the RNA sample.Blotting: It is a second step that involves the blotting of the different RNA sample directly onto the nitrocellulose or nylon filter membrane.Extraction of RNA: Take different samples of the RNA from the different tissues or cells.The identification of RNA by dot blot technique involves the following steps: Autoradiography: Subject the filter membrane to the X-ray film, after which one can visualize the desired gene of DNA.Washing: After hybridization, wash the unbound or free radioactive probe from the filter medium. ![]() A radioactive probe will bind to the target DNA or later hybridize it.
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