Western Blotting

Western blotting is a method or technique of transferring proteins, DNA or RNA, onto a carrier( for example- a nitrocellulose, PVDF or nylon membrane).

There are 3 types of blotting techniques:
  • Northern blotting is used for the detection of RNA.
  • Southern blotting is used for the detection of DNA.
  • Western blotting is used for the detection of Proteins.
Western blotting is widely used analytical technique in molecular biology to detect specific proteins in a sample.It works on the principle of gel electrophoresis (SDS-PAGE).Proteins are separated based on their size on polyacrylamide gel.

Steps in western blotting:

  1. Sample prepration
  2. Gel electrophoresis of protein sample
  3. Protein transfer
  4. Protein staining
  5. Blocking non-specific antibody
  6. Antibody probing
  7. Washing
  8. Protein detection
  9. Digital imaging

Sample prepration: (1) detergent for the lysis of tissue culture. (2)ultrasonication for cell suspension. (3)mechanical homogenization for plant animal tissue. (4)enzymatic digestion for bacterial, yeast, and fungal cells.

Gel electropgoresis is commonly used methos for separating proteins on the basis of size, shape or charge. In this SDS PAGE electrophoresis is used by which protein sample are separated according to there molecular weight.
Electrophoresis

PAGE protocol

  • Sample loading into the wells of electrophoresis equipment
  • loading sample into empty wells
  • place the safety lid on the unit 
  • plug the color coded leads into the jacks in the power supply 
  • run the gel under the appropriate conditions
  • when tracking gel reaches the bottom of the gel turn off the power supply
  • disconnect the leads and removes safety
  • remove the gel from electrophoresis appratus 
  • then proceed to protein transfer

Western blotting procedure:

  • Load and separate protein sample on SDS-PAGE.
  • Electrophoretically transfer fractionated proteins onto membrane.
  • Block the membrane with neutral proteins (BSA or milk casein).
  • Incubate the membrane with primary antibody specific to target protein.
  • Incubate the membrane with HRP(horse radish peroxidase) labeled secondary antibody specific to primary antibody.
  • Incubate the blot with chemiluminescent HRP substrate and expose to X-ray film.
On completion of the separation of proteins by polyacrylamide gel electrophoresis, next step is to transfer the proteins from the gel to solid membrane. usually made up of a chemically inert substanes, such as nitrocellulose or PVDF(polyvinylidene difluoride). the process of transferring proteins from a gel to a membrane while maintaining their relative position and resolutions is known as blotting.
Western blotting


Blocking: For meaningful result the antibodies must bind only to the protein of interest and are not to the membrane. non-specific binding of antibodies can be reduced by blocking the unoccupied sites of membrane with an inert protein or non-ionic detergent. blocking agents should possess greater affinity towards membrane than the antibodies. example of blocking agents are:- bovine serum albumin(BSA), dry milk, gelatine, dilute solution of tween20.

Antibody probing: After blocking the blot is incubated with one or more antibodies. this uses a specific antibody to detect a localize the protein blotted to a membrane. the specificity of antigen of antigen-antibody binding permits the identification of a single protein in a complex sample. the non labeled primary antibody directed against the target protein, and specific labeled secondary antibody binds to the primary antibody. the secondary antibody is conjugated to an enzyme that is used to indicate the location of the protein.the secondary antibodies not only serves as a carrier of the label, but it is also helps to amplify the emitted signals.

Washing: Unbound antibodies can cause high background and poor detection. hence washing the blot removes unbound antibodies from the membrane. a dilute solution of tween-20 in TBS or PBS buffer is commonly used for washing.

Protein detection: After the unbound probes washed away, the western blotting is now ready for detection of the probes that are labeled and bound to the protein of interest. enzyme such as alkaline phosphatase & horse radish peroxidase are widely used in detection of proteins.

Analysis & Imaging: This is the last and major step of western blotting technique. detection of signals using either x-ray film, scanners results in one or more visible proteins bands on the membrane image. the molecular weight of the protein can be estimated by comparision with marker proteins and the amount of protein can be determined as this is related to band intensity.

Applications: 

  • Analysis of IgG fractions purified from human plasma.
  • Diagnosis of HIV by ELISA, involves the western blotting technique.
  • Western blotting technique is also used to detect some forms of lyme disease.
  • Western blotting technique is used in defence test for BSE, which  is commonly known as mad cow disease.
  • Confirmatory test for hepatitis-B involves western blotting technique.
  • This technique is also employed in gene expression studies.  

Limitation of western blotting:

  • Very delicate and time consuming process.
  • Incorrect labeling of protein can happen due to the reaction of secondary antibody.
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