Step VII: Treatment with suitable substrate Secondary antibody (2° Ab) is antibody against primary antibody (anti-antibody) so it can bind with Ag-Ab complex.alkaline phosphatase or Horseradish peroxidase (HRP) is labelled with secondary antibody. The secondary antibody is enzyme labelled.Step VI: Treatment with secondary antibody The primary antibody (1° Ab) is specific to desired protein so it form Ag-Ab complex.So before adding the primary antibody the membrane is non-specifically saturated or masked by using casein or Bovine serum albumin (BSA). Antibodies are also protein so they are likely to bind the nitrocellulose paper.Blocking is very important step in western blotting.In electro-blotting nitrocellulose membrane is sandwich between gel and cassette of filter paper and then electric current is passed through the gel causing transfer of protein to the membrane.For fast and more efficient transfer of desired protein from the gel to nitrocellulose paper electro-blotting can be used.This type of blotting is time consuming and may take 1-2 days The separated protein from gel get transferred to nitrocellulose paper by capillary action. The nitrocellulose membrane is placed on the gel.Protein are negatively charged, so they move toward positive (anode) pole as electric current is applied.The small size protein moves faster than large size protein.The proteins are separated on the basis of electric charge, isoelectric point, molecular weight, or combination of these all.The sample is loaded in well of SDS-PAGE Sodium dodecyl sulfate- poly-acrylamide gel electrophoresis.Tracking dye (bromothymol blue) is also added in sample to monitor the movement of proteins.When sufficient amount of protein sample is obtained, it is diluted in loading buffer containing glycerol which helps to sink the sample in well.The concentration of protein is determined by spectroscopy.To prevent denaturing of protein protease inhibitor is used.This step is also known as tissue preparation. Protein is extracted from cell by mechanical or chemical lysis of cell.Cell lysate is most common sample for western blotting.Treatment with specific substrate if enzyme is alkaline phosphatase, substrate is p-nitro phenyl phosphate which give color.Treatment with secondary antibody( enzyme labelled anti Ab).Blotting: electrical or capillary blotting.Western blotting is also known as immunoblotting because it uses antibodies to detect the protein.

In this method labelled antibody against particular protein is used identify the desired protein, so it is a specific test.Western blotting technique is used for identification of particular protein from the mixture of protein.

Western blot definition