E-Book

E-Book 2nd Congress

  • Western Blotting: a tool for measurement of macromolecules
  • Yasaman Alirezaei,1 Aryan M. Yazdani,2,*
    1. B.S. student of Cellular and molecular biology Islamic Azad University Gorgan
    2. Msc. student of Clinical nutrition Shiraz university of medical sciences


  • Introduction: Blotting techniques are effective for detecting macromolecules such as proteins, DNA and RNAs. Blotting refers to a membrane on which biological molecules, such as proteins and nucleic acids, are immobilized. Western blotting isolates a specific protein from a complex sample or mixture of proteins. It can obtain protein quantity, molecular weight, and post-translational modifications. It is a very sensitive method because of the high affinity of antibodies to their epitope.
  • Methods: Applications of Western Blot 1. Detecting anti-HIV antibodies to diagnose HIV. 2. Quantification of proteins and other gene products in gene expression studies. 3. Evaluating the protein expressions in cells and analysis of protein fractions during protein purification. 4. Analyzing biomarkers like growth factors, cytokines, and hormones. The steps of Western blotting: 1. Preparation of WB buffers: Apart from the lysis buffer needed in sample preparation, other reagents such as loading buffer, running buffer, Coomassie brilliant blue staining solution, and Coomassie decolorization solution are also used to prepare SDS-PAGE. 2. Sample preparation: Western blot sample preparation is performed before SDS-PAGE. Sample extracts can be prepared from cell cultures or tissues by mechanical crushing, such as homogenization and sonication or high-pressure disruption, and then lysing them with an amount of cell lysis buffer to ensure maximum protein extraction. Extraction is performed on ice or at four °C to avoid protein degradation. Appropriate lysis buffers Can be selected based on the desired protein expression site. Protease and phosphatase inhibitors are also added at this stage to protect proteins from digestion by proteases that leak out during sample preparation. 3. Gel electrophoresis: Western blotting involves polyacrylamide gel electrophoresis (PAGE) followed by electrophoretic transfer to a membrane (usually PVDF or nitrocellulose) and an immunostaining method to visualize a specific protein on the blotting membrane. SDS-PAGE is a standard means of separating proteins based on their molecular weight. This technique includes one-dimensional electrophoresis and two-dimensional electrophoresis. PAGE Gel Preparation for One-di Western Blotting SDS-PAGE: A reducing agent is added to denature the protein for most Western blotting routines. 4. Primary antibody incubation for western blotting: After blocking, the membrane is incubated with a primary antibody. The choice of the primary antibody depends on the antigen and its antibodies. Primary antibodies for western blotting include monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). 5. Blocking the membrane and membrane processes: This crucial step prevents the non-specific binding of antibodies to the membrane and is often performed with BSA or nonfat dry milk diluted in TBST/PBST buffer. Regarding these buffers, it is essential to note that TBST with AP (alkaline phosphatase) labeled antibodies are preferred because PBS interferes with it.
  • Results: Methods of western blot detection Direct detection: This method is not widely used. The primary antibody used to detect the antigen on the blot is labeled with a fluorescent dye or an enzyme. Indirect detection: This method is used more often because of its many advantages over the direct detection method. This method adds a primary antibody to bind to the antigen, followed by a secondary antibody labeled against the primary antibody. Labels include biotin, fluorescent probes, such as fluorescein or rhodamine, and enzymatic compounds, such as alkaline phosphatase (AP) or horseradish peroxidase (HRP).
  • Conclusion: Although Western blotting is accepted as a routine method for protein analysis, it has limitations as well as advantages. Advantage: Detection of a very small protein amount (picograms) in a sample. High sensitivity and specificity Most suitable and effective among other techniques for HIV detection. Limitations of western blot: It requires high skill In some cases, antibodies bind to unwanted proteins If no primary antibody is available for the target protein, it cannot be detected. Primary antibodies are expensive The membrane may not retain small proteins.
  • Keywords: Western Blotting-HIV-buffers