Southern Blotting- Northern Blotting- Western Blotting- Eastern blotting

 

Molecular and Medical Biotechnology

Dot- Blotting Techniques

Dot blot is a simple way to test for the presence of a protein of interest (POI) in a sample. The dot blot can also be used for detection of nucleic acids, but for the sake of simplicity.

Dot blot relies on the same principle that many immunological techniques rely on: the recognition and binding of an antigen by an antibody. Briefly, dot blot utilizes a dry nitrocellulose or PVDF membrane that has been "dotted" with sample homogenate (typically a sample volume of ~2uL/dot).

The membrane is then blocked for non-specific binding using a blocking buffer, followed by incubation with a primary antibody specific to the POI for 30 mines to 1 hour at room temperature. This step is then followed by incubation with a secondary antibody (also at room temperature for 30 min to 1 hour) that allows visual detection and quantification of the target protein through methods such as chemiluminescence or fluorescence. Our lab uses the fluorescence based Odyssey system by Liquor for our blots.

This particular technique is usually used for detection and quantification. The technique also provides a quick way to determine if an antibody is non-specific, particularly for a secondary antibody.

As well as there are four types on Dot blot, Southern blotting, Northern blotting, Western blotting and Eastern blotting. The analytical technique that involves the transfer of a specific DNA, RNA or a protein separated on gel to a carrier membrane, for their detection or identification is termed as blotting. The process of transfer of the denatured fragments out of the gel and onto a carrier membrane makes it accessible for analysis using a probe or antibody.

Depending upon the substance to be separated, blotting techniques may be – Southern blot, Northern blot or Western blot which separates DNA, RNA and proteins respectively.

Southern Blot is the analytical technique used in molecular biology, immune genetics and other molecular methods to detect or identify DNA of interest from a mixture of DNA sample or a specific base sequence within a strand of DNA.

The technique was developed by a molecular biologist E.M. Southern in 1975 for analyzing the related genes in a DNA restriction fragment and thus named as Southern blotting in his honor.

 Moreover, moving on principles of southern blot, the process involves the transfer of electrophoresis-separated DNA fragments to a carrier membrane which is usually nitrocellulose and the subsequent detection of the target DNA fragment by probe hybridization. Hybridization refers to the process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target DNA. Since the probe and target DNA are complementary to each other, the reaction is specific which aids in the detection of the specific DNA fragment.

 

1. Extraction and purification of DNA from cells

DNA is first separated from target cells following standard methods of genomic DNA extraction and then purified.

2. Restriction Digestion or DNA Fragmentation

Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments. One or more restriction enzymes can be used to achieve such fragments.

3. Separation by Electrophoresis

The separation may be done by agarose gel electrophoresis in which the negatively charged DNA fragments move towards the positively charged anode, the distance moved depending upon its size.

4. Depuration

Partial depuration is done by the use of dilute HCl which promotes higher efficiency transfer of DNA fragments by it breaking down into smaller pieces.

5. Denaturation

DNA is then denatured with a mild alkali such as an alkaline solution of NaOH. This causes the double stranded DNA to become single-stranded, making them suitable for hybridization. DNA is then neutralized with NaCl to prevent re-hybridization before addition of the probe.

6. Blotting

The denatured fragments are then transferred onto a nylon or nitrocellulose filter membrane which is done by placing the gel on top of a buffer saturated filter paper, then laying nitrocellulose filter membrane on the top of gel. Finally some dry filter papers are placed on top of the membrane. Fragments are pulled towards the nitrocellulose filter membrane by capillary action and result in the contact print of the gel.

7. Baking

The nitrocellulose membrane is removed from the blotting stack, and the membrane with single stranded DNA bands attached on to it is baked in a vacuum or regular oven at 80 °C for 2-3 hours or exposed to ultraviolet radiation to permanently attach the transferred DNA onto the membrane.

8. Hybridization

The membrane is then exposed to a hybridization probe which is a single DNA fragment with a specific sequence whose presence in the target DNA is to be determined. The probe DNA is labeled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye.

9. Washing of unbound probes

After hybridization, the membrane is thoroughly washed with a buffer to remove the probe that is bound nonspecifically or any unbound probes present.

10. Autoradiograph

The hybridized regions are detected auto radio graphically by placing the nitrocellulose membrane in contact with a photographic film which shows the hybridized DNA molecules. The pattern of hybridization is visualized on X-ray film by autoradiography in case of a radioactive or fluorescent probe is used or by the development of color on the membrane if a chromogenic detection method is used.

 

Applications of southern blotting

• Identifying specific DNA in a DNA sample.
• Preparation of RFLP (Restriction Fragment Length Polymorphism) maps
• Detection of mutations, deletions or gene rearrangements in DNA
• For criminal identification and DNA fingerprinting (VNTR)
• Detection and identification of trans gene in transgenic individual
• Mapping of restriction sites
• For diagnosis of infectious diseases
• Prognosis of cancer and prenatal diagnosis of genetic diseases
• Determination of the molecular weight of a restriction fragment and to measure relative amounts in different samples.

 Northern Blotting 

Northern Blotting is a technique used for the study of gene expression. It is done by detection of particular RNA (or isolated mRNA).  MRNA is generally represented as 5% of the overall RNA sequence. This method reveals the identity, number, activity, and size of the particular gene. This blotting technique can also be used for the growth of a tissue or organism. In different stages of differentiation and morphogenesis the abundance of an RNA changes and this can be identified using this technique. It also aids in the identification of abnormal, diseased or infected condition at the molecular level. The northern blot technique was developed in 1977 by James Alwin, David Kemp and George Stank at Stanford University. The technique got its name due to the similarity of the process with Southern blotting. The primary difference between these two techniques is that northern blotting concerns only about RNA.

Figure;1

 As all normal blotting technique, northern blotting starts with the electrophoresis to separate RNA samples by size. Electrophoresis separates the RNA molecules based on the charge of the nucleic acids. The charge in the nucleic acids is proportional to the size of the nucleic acid sequence. Thus the electrophoresis membrane separates the Nucleic acid sequence according to the size of the RNA sequence. In cases where our target sequence is an mRNA, the sample can be isolated through oligo cellulose chromatographic techniques, as mRNA are characterized by the poly (A)-tail. Since gel molecules are fragile in nature, the separated sequences are transferred to the nylon membranes. The selection of nylon membrane is contributed to the factor that nucleic acids are negatively charged in nature. Once the RNA molecules are transferred it is immobilized by covalent linkage. The probe is then added, the probe can be complementary an ssDNA sequence. Form amide is generally used as a blotting buffer as it reduces the annealing temperature.

 Procedure

1.     The tissue or culture sample collected is first homogenized. The samples may be representative of different types of culture for comparison or it can be for the study of different stages of growth inside the culture.

2.     The RNA sequence is separated in the electrophoresis unit an agarose gel is used for the purpose of the nucleic acid separation.

3.     Now the separated RNA sequence is transferred to the nylon membrane. This is done by two mechanisms capillary action and the ionic interaction.

4.     The transfer operation is done by keeping the gel in the following order. First, the agarose gel is placed on the bottom of the stack, followed by the blotting membrane. On top of these paper towels a mild weight (glass plate) is placed. The entire setup is kept in a beaker containing transfer buffer.

5.     RNA transferred to the nylon membrane is then fixed using UV radiation.

6.     The fixed nylon membrane is then mixed with probes. The probes are specifically designed for the gene of interest, so that they will hybridize with RNA sequences on the blot corresponding to the sequence of interest.

7.     The blot membrane is washed to remove unwanted probe

8.     Labeled probe is detected by chemiluminescence or autoradiography. The result will be dark bands in x ray film.

Western blotting

A western blot is a laboratory method used to detect specific protein molecules from among a mixture of proteins. This mixture can include all of the proteins associated with a particular tissue or cell type. Western blots can also be used to evaluate the size of a protein of interest, and to measure the amount of protein expression. This procedure was named for its similarity to the previously invented method known as the Southern blot.

Figure;2

The first step in a western blot is to prepare the protein sample by mixing it with a detergent called sodium dodecyl sulfate, which makes the proteins unfold into linear chains and coats then with a negative charge. Next, the protein molecules are separated according to their sizes using a method called gel electrophoresis. Following separation, the proteins are transferred from the gel onto a blotting membrane. Although this step is what gives the technique the name "western blotting," the term is typically used to describe the entire procedure.

Once the transfer is complete, the membrane carries all of the protein bands originally on the gel. Next, the membrane goes through a treatment called blocking, which prevents any nonspecific reactions from occurring. The membrane is then incubated with an antibody called the primary antibody, which specifically binds to the protein of interest. Following incubation, any unbound primary antibody is washed away, and the membrane is incubated yet again, but this time with a secondary antibody that specifically recognizes and binds to the primary antibody. The secondary antibody is linked to a reporter enzyme that produces color or light, which allows it to be easily detected and imaged. These steps permit a specific protein to be detected from among a mixture of proteins.

Eastern Blotting

Eastern blotting is a simple technique in which the phospholipids and glycosphingo lipids from a high- performance thin layer chromatography (HPTLC) plate is transferred to a poly vinyl-difluoride (PVDF) membrane. It is also called TLC blotting. Most of the lipids developed on HPTLC plate is blotted quantitatively. Detection is done by either chemical or immunological staining. Purification of lipids is also possible in this method. Determination of lipid structure can also be carried out by coupling with mass spectrometry. Identification of enzymes and ligands of microorganisms also can be carried out TLC blotting / MS technique.

In signal transduction process lipids and their metabolites have an important part. These lipids are difficult to handle as secondary messengers in aqueous phase as they form micelle/ liposomes. They behave as large molecules with multiagency. The size of these large molecules vary with method of preparation and experimental conditions. Separation of the proteins which react specifically with particular lipids is exceedingly difficult. The challenges are countered by immobilizing the lipid to a solid phase. This immobilized lipid and the membrane mimics a biological membrane and provides convenient experimental conditions. It separates proteins which do not react with the immobilized lipids. Lipid purifying is difficult limiting lipid research techniques. Column chromatography coupled it ion exchange and repeated silica bead columns have been used to purify lipid molecules. This process is time consuming. It requires a skillful techniques and use of toxic organic solvents.

TLC is the mostly used technique in detection, separation and monitoring of phospholipids and glycosphingolipids and some artificial chemicals and their metabolites.  TLC procedure with monoclonal antibodies is used in the structural analysis of glycosphingolipids. Silica gel used in the HPTLC plate has its own limitation, when to try to exchange the lipid from HPTLC plate there is a risk of silica gel contamination and sloughing off effect of silica gel during treatment is also a problem.  This limitations can be overcame if there is a transfer of lipids from HPTLC plate to a membrane having hydrophobic properties. Good separation of lipids takes place on HPTLC plate and if they are immobilized on a solid surface lipid studies can be made easier. Thus the attempt of lipid from HPTLC plate to the plastic membrane started. Nitrocellulose membrane were also used for the transfer but the transfer efficiency was poor and reproducibility factor was low. Various plastic membranes were tested and finally PVDF membrane suited the best. PVDF membrane is very stable against heating and various organic solvents. Also, the retaining efficiency of lipids on the PVDF is high.  Here is a procedure used for complex lipids.

 Eastern blotting is given by Bogdanovian. This method is used to identify carbohydrate epitopes including glycol conjugates and lipids. Mostly blotted proteins after transferring onto the membrane are analyzed for PTMs by using a probe and hence identify carbohydrates and lipids. It involves the following steps:

•   Firstly, targeted molecules are vertically separated by using gel electrophoresis.
• Then, these separated molecules are transferred horizontally on the nitro cellulosic membrane. After that primary antibody is added to the solution. These antibodies are responsible for recognizing a specific amino-acid sequence. Then wash it to remove unbound primary antibody and add labelled secondary antibody.
•  These labelled probes confirm the molecule of interest.

 

i) Detection of protein modification.

ii) Used for binding studies by using various ligands

iii) Used to purify various phospholipids.

Conclusion

Different blotting is used to detect different type of macromolecules such as southern blotting is used for DNA analysis, western blotting is for protein analysis, northern blotting is for RNA analysis and eastern for carbohydrate detection. The remaining of this article is focus on different techniques and applications used in particular blotting.