Friday, 2 April 2021

Western blotting (Immunoblotting) : Working Principle, Steps & Analysis

Western blotting : Working Principle, Steps & Analysis


 Blot refers to the membrane on which biological molecules such as proteins and nucleic acids are immobilised. The process of transferring macromolecules from a gel to a membrane followed by their detection on the membrane is known as blotting. Western blotting is a widely used technique for the detection and analysis of proteins.

When the macromolecule involved is DNA the technique is known as Southern blotting. Southern is the last name of the scientist who first blotted DNA sir Edwin Melo southern. By analogy blotting involving RNA is known as northern blotting and for protein this technique is known as western blotting.

Western blotting is also known as immunoblotting, this is because antibody probes are used for the detection of the target protein on the membrane.
  There are five main steps involved in Western blotting these are
-  Protein gel electrophoresis
-  Protein transfer
-  Blocking
-  Antibody probing and
-  Detection

Step 1). Protein gel electrophoresis

The first step in Western blotting involves separation and characterization of proteins by Gel electrophoresis.

  Suppose we have three protein samples. Each of these samples contains thousands of proteins. Our aim is to detect presence of a specific protein in these samples. These protein samples are separated into their component proteins by gel electrophoresis.

  Most widely used technique for protein electrophoresis is sodium dodecyl sulfate polyacrylamide gel electrophoresis abbreviated as SDS page. This technique sets proteins according to their molecular weights.

   Another important point is that all the separated proteins in the gel have uniform negative charge. This is because of sodium dodecyl sulfate Which coats the proteins. So after the sds-page the band's represents proteins present in each sample. The left side of the gel is represent in the molecular weight markers.

Step 2). Protein transfer

   Once proteins have been separated by SDS-page, next step is to transfer these proteins from the gel to a suitable membrane. The membranes used in Western blotting are those having high affinity for proteins.

  They have excellent protein binding and retention capabilities. Most commonly used membranes in Western blotting are nitrocellulose and polyvinylidene difluoride abbreviated as PVDF. Protein transfer is done for further detection and analysis of separated proteins.

   The membranes are thinner than gels. Therefore when proteins bind to these membranes, their epitope saw binding sites are easily accessible to the antibodies.

Electrophoretic Transfer

  Method used to transfer proteins from gel to membrane is known as electrophoretic transfer.
In this method electric current is used to elude proteins from gel and transfer them to membranes. The gel and the membrane are placed in the electrophoresis chamber such that, gel is at the side of negative electrode and membrane is at the side of positive electrode.

   Proteins in the gel are negatively charged so they move out of the gel and migrate towards the positive electrode.

Transfer sandwhich

  For protein transfer a stack or transfer sandwich of gel and membrane is prepared first. This sandwich also consists of sponge and filter paper the arrangement of sandwich is like this " sponge, filter paper, gel, membrane, filter paper again followed by sponge".

Filter paper provides a uniform flow of transfer buffer through the gel. This facilitates the movement of proteins out of the gel and on to the membrane.
  The sponge maintained the proper pressure during the transfer.

  This sandwich of gel and membrane is held inside a non-conducting cassette, which keep gel on the membrane in close contact. This complete setup is kept entirely submerged under transfer buffer. Within a buffer tank the placement of cassette is such that gel is at the side of negative electrode, membrane is at the side of positive electrode.

   When electric current is applied, the negatively charged proteins move from gel and traveled toward the membrane. Which is at the side of positive electrode.

   One very important component of transfer buffer is methanol. Methanol helps to increase the binding of proteins to the membrane. This is because during the transfer of protein from gel to membrane methanol removes SDS from proteins.

  At the end of this step all the proteins from gel moved to the membrane and become tightly attached to it. so we have a membrane with copy of band pattern from gel.

Step 3). Blocking

   The third step is known as blocking. The membranes have a very high affinity for proteins our next step will be the addition of antibody to find out the presence of target protein.

   Antibodies are also proteins that means they can bind to empty spaces on the membrane, where no protein bands are present. This nonspecific binding of antibodies to the membrane is detrimental to the specificity and sensitivity of the assay. Therefore it is essential to block spaces that are not already occupied by a proteins. So in this step blocking agent is added to the membrane.

   Most common blocking agents used are bovine serum albumin and non-fat milk. These blocking agents will fill all the unoccupied sites on the membrane. Since these blocking agents specifically bind the membrane they will not disturb the already bound proteins on the membrane.

Step 4). Antibody Probing

   After the blocking step the membrane is incubated with primary antibody. Since this antibody is specific to our target protein it will bind to the protein on the membrane. After this excess of primary antibodies are removed by washing.

Step 5). Detection

   Final step of Western blotting is the detection followed by further analysis of the protein. The membrane is now incubated with labeled secondary antibody.

   Use of secondary antibodies are preferred to maximize the sensitivity of the detection. Multiple secondary antibodies can bind to the target primary antibody and this results in the amplification of detection signal.

  So when the membrane is incubated with labeled secondary antibody. These antibodies bind to the primary antibody that is already bound to the target protein on the membrane. Excessive secondary Antibodies are also removed by washing.

  For the secondary antibody label here is an enzyme. Most preferred enzyme for protein detection in Western blotting is horseradish peroxidase (HRP). 
  In the next step the presence of this enzyme is detected by adding a suitable substrate HRP acts on colorimetric or chemiluminescence substrates.

  The membrane is incubated with a colorimetric substrate such as 4-Cholor-1-napthol (4CN). The enzyme in the membrane catalyzes the oxidation of the substrate into an insoluble purple color product, and this purple color is visible on a blot. 

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