One way we can take a molecular census of married vs divorced protein-RNA (or DNA but I’m an RNA person) couples is with an experiment called a “Slot Blot” or a “Dot Blot” or a “Filter-Binding Assay” – the basic idea is you mix protein and RNA, let them reach a steady marriage/divorce rate (binding equilibrium), then separate the couples from the singles & compare. Please use one of the following formats to cite this article in your essay, paper or report: APA. (2020, January 30). Detection of Histone Proteins Using Western Blot Protocol.

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Example protocols:

If purified, suspend sample in PBS or TBS, or other appropriate buffer. Cell lysates or extracts also may be applied to the membrane or filter for detection of cellular proteins.If using a filtration manifold, dilute sample to 300-500 µl for a concentration of 1-10 µg/spot, then carry out serial dilutions of the sample to determine the optimal concentrations for subsequent binding assays. If not using a manifold, volumes of 2-5 µl should be used.

If using a filtration manifold to apply samples, float membrane in deionized water until completely wet, then soak in PBS or TBS until use. If spotting without a manifold, membrane should be left dry.

Apply sample to membrane directly with a micropipette, or use a filtration manifold. For purified samples, apply 1-10 µg/spot or well. Sample should be diluted in PBS, TBS or other buffer.

Multi-well filtration manifold

Place 2 sheets filter paper, prewet in transfer buffer, on the filter support plate of the Manifold device. Place membrane on top of the filter paper and clamp the sample well plate into place. Apply low vacuum (enough to pull buffer through at approximately 1 mL/minute) and wash each well with 500 µL of sample buffer. While the vacuum is applied, add sample to the well and filter through. After sample has been applied to the membrane, wash with 500 µL of sample buffer. For best results, suspend sample in 300-500 µL of buffer. Unclamp and remove the sample well plate, taking care to leave the membrane on the filter paper. Remove the membrane from the Manifold device.

Direct sample application

If a Manifold device is not used, apply sample to the membrane placed on top of 2 sheets of dry filter paper in 2-5 µL aliquots. If larger sample volumes are used, allow sample spot to dry prior to subsequent 2-5 µL aliquot addition.Note: If using a filtration manifold, low molecular weight samples should be filtered through slowly to prevent passage through the medium, or applied without vacuum.

Follow blocking and detection procedures as indicated for the NitroBind membrane.

Blotting is primarily used in molecular biology. It is used to identify proteins and nucleic acids for diagnostic purpose. Specifically, blotting is used for identifying biomolecules like DNA, mRNA, and protein during gene expression.

DNA and RNA molecules need to undergo biochemistry analysis and they are separated using the blotting method.

Picture : A helpful mnemonic for students to remember the relation between various blotting methods and its uses

Blotting is done by enabling the mixture of molecule pass through a gel that separates the molecules based on their molecular size. The molecules being tested are hard-pressed against a membrane which will then transfer the molecule from the gel onto the suitable membrane through capillary action.

One of the commonly used blotting procedures is Southern blotting. This procedure was introduced in 1975 by Edwin Southern; thus, where the name was taken. What is Southern blotting? It is a blotting method used to detect a particular sequence of DNA in a DNA sample. There are other subtypes such as Northern blotting, Western blotting, South-Western blotting, and Eastern blotting. ^{(1, 2, 3, and 4)}

Image 1: The Southern blot procedure as shown in the image above.
Picture Source: thescienceinfo.com

What does Southern blotting reveal?

A southern blot analysis reveals the following:

• Identity of the DNA
• Size of the DNA
• Abundance of the DNA ^{(4, 5)}

The Southern blotting method is a classic technique that separates DNA fragments according to their size through electrophoresis. They will be transferred to a membrane, hybridize, washed, and lastly, detecting the labeled DNA band.

How much DNA is needed for Southern blotting?

The amount of DNA needed varies depending on the size and specific activity of the probe.

What is the principle of Southern blotting?

Southern blotting is a restriction fragment length polymorphism. It is a hybridization method for identifying the size of DNA from a mixture of other similar molecules. Basically, Southern blotting separates DNA fragments by gel electrophoresis. The DNA fragments are identified using a labeled probe hybridization. ^{(5, 6, and 7)}

What are the steps in Southern blotting?

There are a series of steps that need to be strictly followed when performing Southern blotting. They are the following:

Image 2: The first and second step of southern blot method.
Picture Source: slideplayer.com

#1 – Restriction Digest

A restriction enzyme is used to fragmentize the DNA. The DNA is cut at a specific site generating a fragment. The DNA fragments obtained by restriction digest are amplified by PCR. ^{(7, 8)}

#2 – Gel electrophoresis

Once the DNA fragments are obtained, the next step is separating the DNA fragments using gel electrophoresis.

Image 3: The denaturing of DNA.
Picture Source: slidesharecdn.com

#3 – Denaturation

An SDS gel is needed in this step. Once electrophoresis is done, the SDS gel is soaked in either acid like HCl or alkali like NaOH. The purpose is to denature the fragments of double-stranded DNA. After denaturation, the strands of DNA will separate. ^{(8, 9)}

#4 – Blotting

This is where the actual bloating takes place. The DNA’s separated strands are transferred to a positively charged nylon membrane through the process of blotting.

Blotting process is done so as to determine the nature of DNA

#5 – Baking and Blocking

The DNA is based on autoclave and fix in the membrane. Casein or Bovine serum albumin is used to treat the membrane. What these chemicals do is they saturate the membrane’s binding site.

#6 – Hybridization with labeled probes

A labeled probe is used to treat the membrane-bound DNA. It has complementary sequences to the gene being studied on. The probe will bind with the complementary DNA on the membrane. ^{(3, 7, and 10)}

#7 – Visualization by Autoradiogram

An autoradiogram is used to visualize the membrane bound DNA that is labeled with a probe. Autoradiogram visualization provides a pattern of bands.

Protein Slot Blot Protocol Definition

Why is Southern blotting important?

Image 5: A southern blot is one of the procedures used in paternity testing.
Picture Source: slideplayer.com

Southern blotting is important as it is useful in the various field of interest such as:

1. It is used in detecting the DNA in a given sample such as DNA fingerprinting in forensics.
2. It is useful in isolating and identifying the gene of interest.
3. In forensics, southern blotting is one of the commonly used procedures, especially in:
   1. Criminal identification
   2. Victim identification
4. It is used for paternity testing.
5. It helps identify gene rearrangement or mutation in a particular sequence of DNA/gene mapping.
6. It is used to diagnose a particular disease caused by genetic defects.
7. It is helpful in identifying infectious agents.
8. It helps in restricting fragment length polymorphism.
9. It has the ability to identify a single gene in a pool of DNA fragments.
10. It helps detect cancer and genetic diseases like sickle cell mutation and monoclonal leukemia. ^{(1, 3, 5, and 8)}

How is Southern blotting used in forensics?

Forensic laboratories used southern blotting method to detect the smallest quantity of DNA, especially in the case of rape, thieves, or identification of parenthood. ^{(7, 8)}

Image 6: Southern blotting is a procedure used in a forensic setting such as in the case of rape and other types of crimes that may require identification of DNA samples.
Picture Source: newyorker.com

Summary

Southern blot is a method commonly used in molecular biology. It has been a widely used technique for over three decades. Through southern blot, researchers can thoroughly understand the fundamentals of molecular biology. The primary purpose of southern blot is to detect a sequence of DNA in a given DNA sample.

Cached

However, the southern blotting method can be quite expensive and extremely technical in nature. Sample-wise, it needs a large quantity of DNA, which is not always favorable in some settings.

Southern Blot : Principle, Protocol (steps) And Uses ...

With the advancement in the field of science and medicine, new DNA testing methods were developed such as PCR. It is better than southern blotting because the procedure involved is very straightforward. It is easy, fast, reliable, and only requires a small volume of DNA. ^{(2, 4, 6, and 9)}

References

1. https://en.wikipedia.org/wiki/Southern_blot
2. https://www.onlinebiologynotes.com/southern-blotting-principle-procedure-application/
3. https://www.thermofisher.com/ph/en/home/life-science/dna-rna-purification-analysis/nucleic-acid-gel-electrophoresis/southern-blotting.html
4. https://askabiologist.asu.edu/southern-blotting
5. https://www.sciencedirect.com/topics/neuroscience/southern-blot
6. https://www.mybiosource.com/learn/southern-blotting/
7. https://www.ncbi.nlm.nih.gov/pubmed/18432697
8. https://www.news-medical.net/life-sciences/What-is-Southern-Blotting.aspx
9. https://www.mun.ca/biology/scarr/Gr12-18.html
10. https://www.nature.com/wls/definition/southern-blot-289

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