SOUTHERN BLOTTING & WESTERN BLOTTING

I. Introduction
I.1. Southern blotting

Southern blotting is a molecular biology technique used to detect and analyze DNA.
Developed by scientist Edward Southern in 1975, this method allows for the
identification of specific DNA fragments within a sample.

 Purpose:
 Detection of a specific DNA fragment:
Southern blotting allows researchers to determine whether a particular DNA fragment is
present in a sample, such as when testing for mutations or disease-associated genes.
 Determination of DNA fragment size:
This technique can provide information about the size of the DNA fragment of interest,
which is useful when identifying the size of a gene, intron, or exon in a complex DNA
sample.
 Studying gene structure:
Southern blotting helps in analyzing changes in DNA structure, such as mutations,
insertions, or deletions. It can also be used to study gene recombination or changes in
genome organization.
 Genetic analysis:
In genetic research, Southern blotting is used to study DNA segments related to
hereditary diseases, identify genes in different species, and examine genetic variations in
genomes.
I.2. Western bloting

Western blotting is a molecular biology technique used to detect and analyze specific
proteins within a complex sample. This technique is primarily employed in protein
research

 Purpose:
 Detection of a specific protein:
Western blotting allows for the identification of whether a target protein is present in the
sample, aiding in the detection of disease-related proteins, such as cancer biomarkers or
other disease markers.
 Determination of protein size:
By analyzing the size of the protein, researchers can assess its integrity or detect
variations in the protein, such as mutant forms or isoforms.
 Protein quantification:
Western blotting can be used for relative quantification of protein expression levels under
different conditions, such as pre- and post-treatment with a drug, or between different
tissues.
 Studying protein interactions:
When combined with other techniques, Western blotting can help investigate protein-
protein interactions, providing insights into cellular regulatory mechanisms and various
biological processes
II. Principle of operation
2.2. Southern blotting

 The principle of southern blotting is similar to the blotting technique involving the
transfer of biomolecules from a membrane to another for detection and identification.

 The DNA to be analyzed is digested with restriction enzymes and fractionated by size
by the process of agarose gel electrophoresis.

 The DNA strands are denatured by alkaline treatment and are transferred to a nylon
or nitrocellulose membrane by the blotting process.

 The strands on the membrane are immobilized on the surface by baking or UV

irradiation. The DNA sequences on the membrane can be detected by the process of
hybridization.

 Hybridization reactions are specific as the probes used bind to target fragments
 consisting of complementary sequences.

 The probes used are labeled with different components that can be visualized by
different methods depending on the type of probes used.

2.3. Wesstern blotting

 The principle of western blotting is the interaction between the proteins and the
probes used for the detection of the proteins.
 The proteins used for western blotting are separated by gel electrophoresis to obtain
them on a gel matrix.
 The proteins are then transferred to a nitrocellulose or polyvinylidene fluoride
(PVDF) membrane, where they are immobilized. The transfer of the protein is known
as blotting.
 The protein on the membrane can either be detected by the use of a reporter-labeled
primary antibody directed against the protein or a reporter-labeled secondary
antibody directed at the primary antibody.
 The reporter or probe present on the antibody can be an enzyme that produces a color
reaction or a luminescent signal at the antigen-antibody binding site that produces a
fluorescent signal in the presence of a particular substrate.
 The signal or color generated by the probe requires a detection
system that is appropriate for the signal or intensity generated.
III. Procedure

III.1. SOUTHERN BLOTTING:

1) DNA Sample Preparation

 DNA Extraction: DNA is extracted from cells or tissues (e.g., blood, biopsy tissues).
Various methods can be used to break down the cell and nuclear membranes,
followed by DNA extraction (for example, using chemicals like phenol/chloroform or
commercial DNA extraction kits).
 DNA Quality Check: Measure the concentration and purity of DNA using a
spectrophotometer to ensure the sample is sufficiently clean and contains an adequate
amount of DNA.
 DNA Digestion with Restriction Enzymes: The extracted DNA is then treated with
restriction enzymes. These enzymes cut the DNA at specific sites, generating DNA
fragments of different lengths depending on the genetic sequence of the sample.
 Enzyme Digestion Conditions: The digestion reaction must be carried out under
optimal conditions of temperature and pH to ensure the enzyme functions efficiently.

2) Agarose Gel Electrophoresis

 Agarose Gel Preparation: Dissolve agarose in a buffer solution, then heat it until the
agarose is fully melted. Pour the hot solution into a mold to form the gel. Ethidium
bromide or equivalent stains may be added if the DNA needs to be visualized under
UV light.
 Loading DNA Samples into the Gel: The digested DNA is mixed with loading dye
(a dye for easy visualization) and loaded into the wells of the agarose gel.
 Electrophoresis Run: The gel is placed in an electrophoresis chamber containing
buffer solution. When voltage is applied, the negatively charged DNA fragments will
migrate towards the positive electrode (anode). Smaller DNA fragments move faster
and thus will be found farther along the gel compared to larger fragments.
 Gel Inspection: After the run, the gel can be examined under UV light to observe the
DNA bands if a stain like ethidium bromide is used. This ensures that the DNA has
been properly separated before being transferred onto a membrane.

3) Southern Transfer of DNA to Membrane

 Membrane Preparation: A nitrocellulose or nylon membrane is cut to the size of

the gel and soaked in the transfer solution (typically an alkaline solution to denature
the DNA).
 DNA Denaturation: The DNA on the gel is usually denatured using an alkaline
solution (NaOH) to separate the double-stranded DNA into single strands. This step
is crucial as the probe will hybridize with the single-stranded DNA.
 Transfer Process: There are two main methods to transfer DNA from the gel to the
membrane:
 Capillary Transfer: The gel is placed on top of a water-soaked filter paper, with the
membrane laid on top of the gel. A stack of absorbent paper is placed on top of the
membrane. Water moves from the bottom up through the gel into the membrane,
carrying the DNA fragments onto the membrane.
 Electroblotting: An electric current is applied to move the DNA from the gel to the
membrane, offering a faster alternative to capillary transfer.
 DNA Fixation on the Membrane: After the transfer, the membrane is treated to fix
the DNA, either by heating (baking the membrane at 80°C) or by UV irradiation to
ensure the DNA is securely bound to the membrane.

4) Hybridization with DNA Probe

 Probe Preparation: The probe is a short DNA or RNA sequence that is

complementary to the target DNA region to be detected. The probe can be labeled
with signaling molecules such as radioactive isotopes (e.g., P-32) or non-radioactive
markers like biotin or fluorescein for detection.
 Hybridization: The membrane containing the DNA is incubated in a hybridization
solution with the probe at a specific temperature. The probe will seek out and bind to
complementary sequences on the membrane. Careful temperature control is essential
to ensure that only perfectly complementary sequences form stable hybrids.
 Washing the Membrane: After hybridization, the membrane is washed with specific
solutions to remove any unbound or non-specifically bound probes. This step helps to
reduce background noise and increase the specificity of the detection method.

5) Signal Detection

 Radioactive Signal: If the probe is labeled with a radioactive isotope (usually P-32),
the membrane is exposed to X-ray film to detect the signal through autoradiography.
DNA bands bound to the probe will appear as dark bands on the film.
 Fluorescent Signal: If the probe is labeled with a fluorescent tag (e.g., biotin,
fluorescein), the signal is detected using a scanner or a fluorescent detection device.

6) Result Analysis

 Comparison of DNA Bands: The DNA bands on the membrane are compared to
control samples or a DNA ladder. If there are mutations or changes in the target gene,
the size or position of the bands will differ from those of the non-mutated sample.
 Mutation Detection: By comparing the DNA bands, the researcher can determine
whether there are mutations, gene amplifications, or gene rearrangements. This helps
in diagnosing genetic disorders or gene-related diseases.

III.2. WESTERN BLOTTING:

1) Protein Sample Preparation

- Cell Lysis:
Use a lysis buffer containing protease and phosphatase inhibitors to prevent protein
degradation. Common additives in lysis buffers include:

 Tris-HCl (50 mM, pH 7.4-8.0): Maintains the proper pH.

 NaCl (150 mM): Adjusts osmolarity.
 NP-40, Triton X-100, or SDS: Detergents to break cell membranes and solubilize
membrane proteins.
 Protease inhibitors such as PMSF (phenylmethylsulfonyl fluoride) or protease
inhibitor cocktails.

- Protein Extraction:

The cell lysate should be centrifuged at high speed (typically 10,000-15,000 g for 10-20
minutes at 4°C) to remove cell debris.

The supernatant containing proteins is collected and stored at -80°C if not used
immediately.

- Protein Quantification:

Measuring protein concentration is necessary to ensure equal amounts of protein are

loaded into the gel. Common methods for protein quantification include:

 Bradford assay: Uses Coomassie Brilliant Blue dye.

 BCA assay: Uses bicinchoninic acid reagent.

Typically, 20-50 µg of protein per well is loaded for Western blotting.

- Protein Denaturation:

 Mix the protein sample with sample loading buffer (Laemmli buffer) containing:
 SDS (Sodium Dodecyl Sulfate): Denatures proteins by coating them with negative
charge.
 β-mercaptoethanol or DTT (Dithiothreitol): Breaks disulfide bonds in proteins.
 Bromophenol Blue: A tracking dye to monitor the electrophoresis process.
 The sample is then heated at 95-100°C for 5 minutes to fully denature the proteins.

2) SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)

- Polyacrylamide Gel Preparation:

 SDS-PAGE gels are prepared with varying concentrations of polyacrylamide
depending on the size of the protein to be separated:
 Large proteins (>100 kDa): 6-8% gel.
 Medium proteins (30-100 kDa): 10% gel.
 Small proteins (<30 kDa): 12-15% gel.
 Stacking gel: Typically has a lower polyacrylamide concentration (4-5%) to help
focus the proteins into a tight band before they enter the resolving gel for separation.

- Electrophoresis:

 After loading the samples into the wells of the gel, electrophoresis is initiated at 80-
100V in the stacking gel and increased to 120-150V once the samples enter the
resolving gel.
 A protein ladder (molecular weight marker) is also loaded into one well to serve as
a reference for determining the size of the proteins after separation.

3) Protein Transfer from Gel to Membrane (Blotting)

- Choosing the Transfer Membrane:

 Nitrocellulose or PVDF membranes are used to capture proteins. PVDF membranes

need to be activated with methanol before use.

- Transfer Methods:

 Wet Transfer: This method is performed in a transfer buffer containing methanol

(20%) and Tris-glycine. Proteins are transferred from the gel to the membrane using
an electric current (typically 100V for 1-2 hours) at 4°C to preserve the integrity of
the proteins.
 Semi-dry Transfer: This method is faster (20-30 minutes) and requires less buffer,
but it may be less efficient for larger proteins (>100 kDa).

- Membrane Blocking

 Blocking Solution:
A blocking solution is used to prevent non-specific antibody binding to the
membrane. Common blocking solutions include:
 5% skim milk in TBS-T (Tris-buffered saline with 0.1% Tween-20).
 1-5% BSA (Bovine Serum Albumin) for cases where antibodies are sensitive to
components in milk.
 The membrane is incubated in the blocking solution at room temperature for 1 hour
or overnight at 4°C.

4) Antibody Incubation

- Primary Antibody:

 The membrane is incubated with a primary antibody specific to the target protein.
The primary antibody can be polyclonal or monoclonal and is typically diluted in the
blocking solution (usually 1:1000 to 1:5000).
 Incubation can last 1-2 hours at room temperature or overnight at 4°C.

- Membrane Washing:

 After incubating with the primary antibody, the membrane is washed multiple times
with TBS-T to remove any non-specifically bound antibodies.

- Secondary Antibody:

 The secondary antibody recognizes and binds to the primary antibody. It is usually
conjugated to enzymes such as HRP (Horseradish Peroxidase) or alkaline
phosphatase.
 The secondary antibody is typically diluted in the blocking solution (1:2000 to
1:10000) and incubated at room temperature for 1 hour.

- Final Washing:

 After incubation with the secondary antibody, the membrane is washed again
thoroughly to remove any unbound secondary antibodies.

5) Signal Detection

- Substrate Preparation:

 If the secondary antibody is linked to HRP (Horseradish Peroxidase), an ECL

(Enhanced Chemiluminescence) substrate is used. The reaction between HRP and
ECL generates light, which can be detected by a chemiluminescent detector or X-ray
film.

- Signal Detection:

 The emitted light is captured on X-ray film or through a CCD camera system.
 For alkaline phosphatase detection methods, substrates like BCIP/NBT produce a
color, and the membrane will display a visible color corresponding to the target
protein.

Result Analysis:

 Protein bands will appear on the membrane. The sizes of the bands are compared to a
protein ladder to determine the molecular weight of the target protein.
 Specialized software can be used to quantify the intensity of the protein bands,
allowing comparison of protein expression levels between different samples.

Important Notes:

 Antibody Optimization: It is necessary to test different concentrations of both

primary and secondary antibodies to find the optimal conditions.
 Loading Control: Constant proteins such as β-actin or GAPDH are commonly used
as internal controls to ensure equal amounts of protein are loaded into the wells.

IV. Comparison
 Similarities:
Sample separation method: separating the molecules through gel electrophoresis.
 Differences:
 Southern blotting Western blotting

Target molecule DNA Protein

Sample DNA extraction enzymatic Protein extraction

preparation digestion

Membrane Nylon Nitrocellulose or PVDF

material ( Polyvinylidene Fluoride )

Probe Nucleic acid probe with Primary antibody

sequence homologous to
target

Probe lable Radiolable, enzyme Enzyme

Detection method X - ray film, CCD camera ( Charge-Coupled

chemiluminescence Device camera), Film, LED or
infrared imaging mechanism

V. Practice applications
V.1. Southern blotting in genetic research
V.2. Western blotting in medicine and protein research
VI. Conclusion
VI.1. Southern blotting
 Advantages:
 Multiple similar sequences in the genome can be analyzed with a single
probe
 Ideal for detecting gene rearrangements if breakpoints are highly variable
 Targets can be scattered over a large genomic region
 Still provides results if a novel partner is involved

Very useful for highly complex samples
 Disadvantages:
  It is labor-intensive
 It is not scalable (only a limited number of samples can be processed
simultaneously)
 Rarely, variants in restriction enzyme cutting sites can lead to atypical
results
 Requires large amounts of extracted DNA
 Process is time-consuming and cannot be accelerated
VI.2. Western blotting
 Advantages:
• Sensitive test
• Nice, clean easily interpretable output
• Thousands of commercially available antibodies
• Tests denatured proteins, so changes in functional states can be detected
 Disadvantges:
• Not quick, slightly labor-intensive
• Tissue must be homogenized
VII. References
1. https://www.aatbio.com/resources/application-notes/
southern-blot-principles-example-workflow-applications
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/
3. https://microbenotes.com/southern-blot/#principle-of-
southern-blot
4. https://microbenotes.com/western-blot/#principle-of-
western-blot
5. https://byjus.com/neet/difference-between-northern-
southern-and-western-blotting/
6. https://www.labmanager.com/southern-vs-northern-vs-
western-blotting-techniques-854
7. https://www.sinobiological.com/category/what-is-wb
8. Molecular Biomethods Handbook - 2nd Edition ( Edited by
John M. Walker Professor Emeritus School of Life Sciences
University of Hertfordshire Hatfield, Hertfordshire, UK
Ralph Rapley School of Life Sciences University of
Hertfordshire Hatfield, Hertfordshire, UK )