Overview of Blotting

Maria Cristina Negritto1, Glenn M. Manthey2

1 Pomona College, Claremont, California, 2 City of Hope, Beckman Research Institute, Duarte, California
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 8.1
DOI:  10.1002/9780470089941.et0801s00
Online Posting Date:  October, 2008
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Abstract

Blotting techniques are among the most common approaches used in a molecular biology laboratory. These techniques, Southern, northern, and immunoblotting, are applicable to a variety of macromolecules including DNA, RNA, and protein, respectively. Each of the techniques are dependent on the ability to resolve the individual macromolecules in a size‐dependant manner, transfer the molecules to a solid support, and finally use a defined probe to detect the specific molecule of interest. The utilization of the blotting technology over the last 30 years has been instrumental to the elucidation of many fundamental biological processes. The continued use of blotting technology holds promise for even greater discovery over the next 30 years.

Keywords: Southern; Northern; Western; Probe; Hybridization; antibody; membrane; blotting; protein blot; DNA blot; RNA blot

     
 
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Table of Contents

  • General Overview
  • General Considerations
  • Southern and Northern Blotting
  • Immunoblotting
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Figures

  •   FigureFigure 8.1.1 Blotting Procedure. General overview of the steps involved in a blotting procedure that are common to nucleic acid blotting (Southern and northern) and protein blotting (western or immunoblotting). (1) Separation of the molecules by gel electrophoresis on either an agarose (DNA or RNA) or a SDS‐polyacrylamide gel (protein). (2) Resolved molecules are transferred to a membrane maintaining the same pattern of separation they had on the gel. (3) The blot is treated with blocking agents, such as proteins (BSA) or detergents that bind to unoccupied sites on the membrane. This is depicted as a gray background. (4) A specific probe that binds to the protein or nucleic acid sequence of interest is incubated with the blot. In the case of a Southern or northern blot the probe consists of a complimentary DNA or RNA sequence. For an immunoblot, the probe consists of a primary antibody that recognizes a particular protein or epitope. (5) Detection step. When using a radioactively labeled probe, the signal is detected by X‐ray film or phosphorimager, resulting in the banding pattern depicted in step 6. Nonradioactive probes can utilize a reporter enzyme directly conjugated to the probe or a labeling moiety that is then detected by a specific antibody conjugated to a reporter enzyme. (6) The reporter enzymes are then presented with colorimetric, fluorogenic, or chemiluminescent substrates that produce signals, which can be detected as a colored product (analyzed visually), as a fluorescent precipitate (detected with a camera after excitation), or as a compound that emits light during its decomposition (detected with X‐ray film or a cooled CCD camera).
  •   FigureFigure 8.1.2 Gel documentation and standard curve. (A) Photograph of a 14 × 14–cm 0.8% agarose gel with samples of restricted yeast genomic DNA, stained with ethidium bromide and aligned with a fluorescent ruler. (B) Standard curve of the migration distances of the molecular weight standards resolved on the agarose gel and plotted as a function of size (bp) versus distance migrated from the origin (cm).
  •   FigureFigure 8.1.3 Structures of the five major classes of secreted antibody. Light chains are shown in light gray; heavy chains are shown in dark gray. Circles denote areas of glycosylation. The polymeric IgM and IgA molecules contain a polypeptide known as the J chain. The dimeric IgA molecule shown includes the secretory component. Reproduced with kind permission from Coico et al. ().

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Literature Cited

Literature Cited
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