What’s the difference between a Southern, Northern, and Western Blot?

blot-technique-comparisons

Different blotting techniques are used to identify unique proteins and nucleic acid sequences. Southern, northern, and western blot protocols are similar, and begin with electrophoretic separation of protein and nucleic acid fragments on a gel, which are then transferred to a membrane (nitrocellulose membrane, polyvinylidene difluoride (PVDF) membrane, etc.) where they are immobilized. This enables radiolabeled or enzymatically labeled antibody or DNA probes to bind the immobilized target, and the molecules of interest may then be visualized with various methods. Blotting techniques are selected based on the target molecule: DNA, RNA, or protein. (Figure 1, Table 1).

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Southern Blot

Southern blots are used to determine the identity, size, and abundance of specific DNA sequences. The southern blot protocol begins with DNA extraction from the cells or tissues, which is then enzymatically digested to produce DNA fragments. The fragments are separated by size on an agarose or polyacrylamide gel via electrophoresis. Smaller fragments will migrate farther on the gel than larger ones. Following electrophoresis, the DNA on the gel is transferred to a nylon membrane. The membrane is incubated with a nucleic acid probe that has a sequence homologous to the target sequence and is labeled with radioactivity, fluorescent dye, or an enzyme capable of generating a chemiluminescent signal. Hybridization of complementary sequences occurs during incubation, and the unhybridized probe is removed by washing with buffer. The fully hybridized labeled probe molecules will remain bound to the blot. Detection methods differ based on the probe label; radiolabeled probes are visualized with X-ray film or phosphorimaging, and enzymatically labeled probes are visualized with chemiluminescent substrate.  

Southern blot protocol

  1. DNA isolation
  2. Restriction digestion: digest the DNA with a restriction enzyme, and if necessary, concentrate digested DNA.
  3. Gel electrophoresis: prepare an agarose gel and either TAE or TBE buffer (buffer selection will depend on the duration of the run and the size of the DNA fragments). Load samples into wells and include a DNA molecular weight marker. Run the gel.
  4. Transfer:
    • Place the gel in a container with denaturing solution, and wash twice for 15 minutes on a shaker.
    • Rinse with water, then wash with neutralization solution.
    • During the previous step, begin to prepare Whatman paper and nylon membrane for the transfer.
    • Assemble the transfer apparatus with the membrane, Whatman paper, and gel and transfer in SSC or SSPE buffer.
    • When transfer is complete, cross-link DNA in a cross-linker, then rinse the membrane.
  5. Pre-hybridization (blocking):
    • Blocking reduces non-specific binding to the membrane. Prepare the pre-hybridization solution and add sample DNA. Remove the blot from the cross-linker, add the pre-hybridization solution and incubate.
  6. Hybridization:
    • Prepare the probe mixture (a complementary DNA strand) and buffer.
    • Remove the pre-hybridization solution and incubate the blot with the probe (incubation times will vary depending on the application).
    • Following incubation, perform a low-stringency wash followed by a high-stringency wash to refine the DNA.
  7. Probe detection:
    • Rinse the membrane, transfer to a container with blocking solution and incubate.
    • Discard blocking solution, replace with antibody solution and incubate.
    • Discard antibody solution, wash the membrane.
  8. Follow manufacturer directions for chemiluminescent detection.

 

Northern Blot

Northern blots are used to determine the identity, size, and abundance of specific RNA sequences. Northern blot protocols begin with RNA isolation, and separation techniques vary depending on RNA size. Large RNAs are separated by electrophoresis on a formaldehyde agarose gel or glyoxal agarose gel, which prevents normal base paring and maintains RNA in a denatured state. Small RNAs are separated on a denaturing (urea) polyacrylamide gel.  The RNA is then transferred from the gel to a nylon membrane which is then incubated with a radioactively or nonisotopically labeled RNA, DNA, or oligodeoxynucleotide probe. The unhybridized probe is removed by washing with buffer. Radiolabeled probes are visualized with X-ray film, and enzymatically labeled probes are visualized with chemiluminescence.

Northern blot protocol

  1. RNA isolation
  2. Electrophoresis:
    • For a formaldehyde agarose gel: prepare the gel and insert the gel tray into the apparatus. Fill with MOPS buffer, load the samples and include a molecular weight marker. Run the gel, then trim the gel prior to blotting.
    • For a glyoxal agarose gel: prepare the gel and insert the gel tray into the apparatus. Fill with MOPS buffer, prepare samples and load into wells along with RNA ladder.
    • For a denaturing polyacrylamide gel: cast the gel, and mount it in the electrophoresis unit. Prepare samples, load into the gel, and run with TBE running buffer.
  3. Transfer:
    • For a formaldehyde agarose gel or glyoxal agarose gel: wash the gel in SSC, then assemble the transfer unit with the gel, filter paper, and nylon membrane. When transfer is complete, place the membrane in a UV cross-linker.
    • For a denaturing polyacrylamide gel: assemble the transfer unit including gel, filter paper, and nylon membrane ensuring they are flooded with TBE. When transfer is complete, place the membrane in a UV cross-linker to fix the RNA to the membrane.
  4. Pre-hybridization (blocking):
    • Pre-hybridize the membrane in hybridization solution.
  5. Hybridization:
    • Add probe to the hybridization solution and incubate.
    • Wash the membrane in low-stringency washes to remove hybridization solution and unhybridized probe, and high-stringency washes to remove partially hybridized molecules.
    • Follow manufacturer directions for chemiluminescent detection.

Western Blot

Direct-detection-labeled-primary-antibody

Western blots are used to determine the identity, size, and abundance of specific proteins within a sample. The western blot protocol begins with  sample lysate preparation from tissue or cell culture and separation on a polyacrylamide gel via electrophoresis. The separated proteins are then transferred to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. The membrane is incubated with a blocking agent to prevent nonspecific binding, followed by incubation with a primary antibody to bind the protein of interest. There are two detection methods, direct and indirect. Direct detection (Figure 2) relies on a labeled primary antibody, whereas indirect detection requires a primary antibody directed against the target protein, and a secondary antibody directed against the immunoglobin class or subclass of the primary antibody’s species (Figure 3). Visualization methods include colorimetric assays in which a colored precipitate is produced, chemiluminescence, and fluorescence.

Indirect-detection-primary-antibody-labeled-secondary-antibody

Western blot protocol

  1. Prepare lysate from cell culture or tissue.
  2. Sample preparation:
    • Determine the protein concentration of each sample with a protein quantification assay (ie. Bradford assay).
    • Add an equal volume of 2X Laemmli sample buffer to each sample.
    • Some samples may need to be reduced or denatured, this is achieved by boiling samples in buffer.
  3. Electrophoresis:
    • Prepare an SDS-PAGE gel, load samples along with molecular weight marker.
    • Run the gel in running buffer.
  4. Transfer:
    • Following electrophoresis, assemble the transfer unit including the gel, PVDF or nitrocellulose membrane, and filter paper.
    • Transfer the proteins to the membrane with transfer buffer.
  5. Antibody Staining (indirect detection):
    • Prepare blocking buffer and incubate the membrane to reduce non-specific binding.
    • Incubate the membrane with primary antibody diluted in blocking buffer.
    • Wash the membrane in TBST, and incubate with conjugated secondary antibody diluted in blocking buffer.
    • Wash the membrane in TBST.
    • Follow manufacturer directions for chemiluminescent detection.