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Friday, August 6, 2021

Blotting method research

Blotting method research:

·        Blot analysis is a strong technique for detecting particular biomolecules in materials with a diverse makeup.

·        Blotting methods are used in conjunction with gel electrophoresis, which is a resolving process for separating DNA, RNA, and proteins.

·        It may be used on biomolecules that can bind their corresponding ligand while adhering firmly to a support material like nitrocellulose, nylon, or paper membrane. Before being transferred to the membrane, the biomolecules of interest are usually sorted by size and/or charge.

·        Edwin M. Southern invented the technique for DNA analysis in 1975. He demonstrated that electrophoretically fractionated DNA restriction fragments may be transferred to a solid substrate (nitrocellulose) and identified as distinct bands after hybridization with a complementary DNA probe.

·        Northern Blotting was coined after the Southern blotting process was applied to RNA. Western blot analysis is the process of transferring proteins to a membrane and detecting them using an antibody probe.

·        All of the techniques involve a stage in which molecules are moved from the gel to a porous membrane, which is usually accomplished by soaking a solution through the gel and membrane with absorbent paper.

·        Molecular hybridization with labelled nucleic acid probes detects particular sequences in the membrane for DNA and RNA. Labeled antibodies are used to detect the proteins.

·        The original procedure was modified to work with radioactive probes labelled with 32P, 3H, 35S, or 125I, for example. Other labelling methods, including as fluorescent and chemiluminescent chemicals, have been developed since then.

Principles in general

All three blotting procedures are quite straightforward, with four distinct steps:

1.      Separation of protein or nucleic acid fragments in a sample by electrophoresis

2.      Immobilization of analytical probe on paper,

3.      Binding of analytical probe to target molecule on paper, and

4.      Visualization of bound probe

Electrophoresis is used to separate molecules in a sample before they are transported to a support medium or membrane that can be handled easily. This immobilises the protein or DNA fragments, allowing for a more accurate replication of the initial separation and easier biochemical examination.

After being transferred to the support medium, the immobilised protein or nucleic acid fragment is localised using probes that selectively attach to the molecule of interest, such as antibodies or DNA. Finally, autoradiography is used to observe the location of the probe that is attached to the immobilised target molecule.

Procedure:

1.      Run the DNA/RNA/Protein gel to the required length.

2.      Use a UV box to visualise the gel.

3.      Soak the gel in 0.2 N HCl until the colour of the bromophenol blue band starts to change. (It generally takes around 10 minutes.) Alternatively, expose for 90 seconds to a UV transilluminator.

4.      Rinse with H2O once more.

5.      Soak for 30 minutes in 0.4N NaOH/0.6M NaCl

6.      Create a transfer pyramid by placing a glass plate on a support in a tray holding 1 litre of 0.5 M NaOH and covering it with two sheets of filter paper (Whatman 3MM or other appropriate paper) so that it contacts the solution on all sides and serves as a wick.

7.      Wet the filter paper with 0.5 M NaOH and gently spin a glass pipette back and forth over the paper to eliminate air bubbles.

8.      Place the filter paper on top of the gel. Place a piece of nylon membrane on the gel that is slightly bigger than the gel and has been prewet in 0.5 M NaOH. 4 sheets of cut-to-size prewet filter paper Between each layer, make sure there are no air bubbles.

9.      Place a 10 cm stack of dry filter paper or paper towels on top of the pyramid, trimmed to size. Place a glass plate on top and a 0.5 kg weight on top.

10.   Mark the orientation of the gel and nylon filter, for example, by cutting off a corner. Wrap strips of parafilm or plastic wrap over the gel to prevent direct contact between the filter paper stack and the wick, which might cause a short-circuit in the transfer solution's flow. Allow 12 hours or more for the transfer to complete.

11.   Dismantles the pyramid with care.

12.   Remove the membrane from the blotter. Remove the gel, whatman, and paper towels from the equation.

13.   Using 2X SSC, rinse the membrane. Using a piece of whatman, blot dry.

14.   Bake for 1 hour in a vacuum oven with the blot sandwiched between two pieces of dry whatman.

Blotting Techniques in Practice

Northern Blotting

Northern blotting allows researchers to compare the expression patterns of a gene across tissues, organs, developmental stages, stress conditions, and pathogen infection.

When contrasted to 'normal' tissue, the method has been used to reveal overexpression of oncogenes and downregulation of tumor-suppressor genes in malignant cells, as well as gene expression in the rejection of donated organs.

Southern Blotting

Southern blotting has a variety of uses in molecular biology, including detecting RFLPs used in the building of genomic maps and identifying one or more restriction fragments that contain a gene or other DNA sequence of interest.

Western Blotting

The western blot (also known as an immunoblot) is a technique for detecting particular proteins in a tissue homogenate or extract sample. The technique was developed in George Stark's Stanford laboratory. W. Neal Burnette coined the term "western blot" to describe the procedure.

 

 

 

 

 

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