Mastering the Art of Blotting: Essential Techniques for Beginners

Mastering the Art of Blotting: Essential Techniques for Beginners


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  • Source: Microbioz India

  • Date: 22 Nov,2023

Various molecular biology applications require skillful blotting techniques mastering.

Here are the basic techniques for beginners:

Southern Blotting (DNA):

Purpose: To identify specific DNA sequences


  1. DNA Digestion: DNA cut by restriction enzymes.
  2. Gel Electrophoresis: Separation of DNA fragments by size using an agarose gel.
  3. DNA Denaturation: Alkaline treatment of the DNA to denature it.
  4. Blotting: Transfer of DNA to a membrane (nitrocellulose or nylon).
  5. Probe Hybridization: Labeled probe binds to the target sequence.
  6. Detection: Labeled bands visualized by autoradiography or chemiluminescence.

Northern Blotting (RNA):

Purpose: Examining levels of RNA expression

  1. RNA Electrophoresis: Separation based on size.
  2. RNA Transfer: Moving from gel to membrane
  3. RNA Fixation: Crosslinking RNA to the membrane.
  4. Hybridization: Probing with a labeled RNA or DNA probe
  5. Detection: Autoradiography and other detection methods.

Western Blotting (Protein):

Purpose: Identify and quantify defined proteins

  1. Protein Separation: Proteins separated via SDS-PAGE
  2. Protein Transfer: Nitrocellulose or PVDF membrane transfers proteins.
  3. Blocking: Blocking agents that prevent nonspecific binding
  4. Antibody Incubation Probe with specific primary and secondary antibodies.
  5. Detection Use chemiluminescence or fluorescence for visualization.

Dot Blotting:

Purpose: Quick analysis of protein or nucleic acid samples.

  1. Sample Application Directly spotted on membrane.
  2. Blocking Preventing non-specific binding.
  3. Antibody or Probe Binding: Incubate with specific antibodies or probes.
  4. Detection Visualize the spots using appropriate detection methods.

Techniques for Optimization:

  1. Blocking: Nonspecific binding should be avoided by complete covering of the surface.
  2. Washing: Cut down on the number of washes to lower background.
  3. Detection Methods: Selecting the correct method depending on sensitivity and ease of use.
  4. Positive and Negative Controls: Each step must have a control to ascertain its success.


  1. Record Keeping Experimental conditions, reagents, and results should be documented in detail.
  2. Image Documentation Capture images of blots for analysis and future reference.


  1. Artifact Identification Smearing or incomplete transfers are some common problems you will learn to recognize.
  2. Optimization The adjustment of conditions in a systematic manner aims at solving problems.

Beginners who understand and practice these basic blotting techniques can establish a firm footing for accurate and reproducible molecular biology experiments.

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