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Blotting Techniques
Blotting techniques are used to detect and analyze three types of biological macromolecules: DNA, RNA and proteins.
This technique is accomplished by:
- Separation using electrophoresis
- Transfer to a membrane
- Detection with a labeled probe
Types of blotting
The 4 basic types of blotting are
- Southern
- Northern
- Western
- Eastern (rarely used in clinical setting)
PRINCIPLE
- The blotting methods are fairly simple and usually consist of four separate steps: electrophoretic separation of protein or of nucleic acid fragments in the sample; transfer to and immobilization on paper support; binding of analytical probe to target molecule on paper; and visualization of bound probe.
- Molecules in a sample are first separated by electrophoresis and then transferred on to an easily handled support medium or membrane.
- This immobilizes the protein or DNA fragments, provides a faithful replica of the original separation, and facilitates subsequent biochemical analysis. After being transferred to the support medium the immobilized protein or nucleic acid fragment is localized by the use of probes, such as antibodies or DNA, that specifically bind to the molecule of interest. Finally, the position of the probe that is bound to the immobilized target molecule is visualized usually by autoradiography.
SOUTHERN BLOTTING
- It is a classic technique that involves separating DNA fragments based on size via electrophoresis, transferring them to a membrane, hybridization with a labeled sequence-specific probe, washing, and finally detection of labeled DNA band(s).
- The method is named after its inventor, the British biologist Edwin Southern.
- DNA finger printing is an example of southern blotting. Used for paternity testing, criminal identification, victim identification. To isolate and identify desire gene of interest.
- Normal chromosomal or gene rearrangement can be studied using this technique.
- Can be used to find similar sequences in other species or in the genome by decreasing the specificity of hybridization.
- Moreover it is used to identify a specific region that uses many different restriction enzymes in a restriction mapping.
- Also it is used to determine which recognition site has been altered due to a single nucleotide polymorphism that changes a specific restriction enzyme.
Limitations
- Compared to other tests, southern blot is a complex technique that has multiple steps and these steps require equipment and reagents that are expensive.
- High quality and large amounts of DNA are needed.
- Southern blot is a time consuming method and can only estimate the size of the DNA since it is a semi-quantitative method.
- It cannot be used to detect mutations at base-pair level.
| STEPS |
|---|
| Step 1: DNA digestion |
| Step 2: Gel electrophoresis |
| Step 3: Blotting |
| Step 4: Probe labeling |
| Step 5: Hybridization & washing |
| Step 6: Detection |
NORTHERN BLOT
- Northern blot is a laboratory analysis method used to study RNA.
- Specifically, purified RNA fragments from a biological sample (such as blood or tissue) are separated by using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments to move faster than larger fragments
- The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University.
- The first step in a northern blot is to denature, or separate, the RNA within the sample into single strands, which ensures that the strands are unfolded and that there is no bonding between strands. T
- The RNA molecules are then separated according to their sizes using a method called gel electrophoresis.
- RNA is transferred from the gel onto a blotting membrane. (Although this step is what gives the technique the name "northern blotting," the term is typically used to describe the entire procedure.)
- Once the transfer is complete, the blotting membrane carries all of the RNA bands originally on the gel.
- Next, the membrane is treated with a small piece of DNA or RNA called a probe, which has been designed to have a sequence that is complementary to a particular RNA sequence in the sample; this allows the probe to hybridize, or bind, to a specific RNA fragment on the membrane.
- In addition, the probe has a label, which is typically a radioactive atom or a fluorescent dye.
- Thus, following hybridization, the probe permits the RNA molecule of interest to be detected from among the many different RNA molecules on the membrane.
Disadvantage
- A problem in northern blotting is often sample degradation by RNases (both endogenous to the sample and through environmental contamination), which can be avoided by proper sterilization of glassware and the use of RNase inhibitors such as DEPC (diethylpyrocarbonate).
- The chemicals used in most northern blots can be a risk to the researcher, since formaldehyde, radioactive material, ethidium bromide, DEPC, and UV light are all harmful under certain exposures.
Advantages
- The detection of RNA size, the observation of alternate splice products, the use of probes with partial homology, the quality and quantity of RNA can be measured on the gel prior to blotting, and the membranes can be stored and reprobed for years after blotting.
WESTERN BLOT
- The western blot (alternatively, immunoblot) is used to detect specific proteins in a given sample of tissue homogenate or extract.
- The method originated from the laboratory of George Stark at Stanford. The name western blot was given to the technique by W. Neal Burnette
- Steps involved in western blot, including sample preparation, gel electrophoresis, proteins transfer, blocking, antibody incubation, and proteins detection and visualization.
| STEPS |
|---|
| Sample preparation |
| Gel electrophoresis |
| Proteins transfer |
| Blocking |
| Antibody incubation |
| Proteins detection |
| Visualization |
Advantages
- Assessing genome data can tell you if the organism in question has the potential to produce certain proteins.
- Providing information on the location and fate of the protein in question
- Western blotting on time course experiment samples can provide information about protein level changes over time, for example during different cell cycle phases.
- Overall, western blotting is not considered a particularly quantitative technique compared to the likes of PCR. However, the direct detection of proteins it offers means it has found great utility in confirmatory disease diagnoses.
- Biomarkers of non-infectious disease
- Western blot has been used to confirm cases of diseases such as human immunodeficiency virus (HIV),Lyme disease, bovine spongiform encephalopathy (BSE)40 and aspergillosis. However, it is time consuming compared to techniques such as PCR and has been replaced by alternative assays in a number of cases including HIV testing.
EASTERN BLOTTING
- It is a technique to detect protein post translational modification and is an extension of the biochemical technique of western blotting.
- Proteins blotted from two dimensional SDS-PAGE gel on to a PVDF or nitrocellulose membrane are analyzed for post-translational protein modifications using probes specifically designed to detect lipids, carbohydrate, phosphomoieties or any other protein modification.
- Most of the proteins that are translated from mRNA undergo modifications before becoming functional in cells. The modifications collectively, are known as post-translational modifications (PTMs).
