Lab technicians utilize a variety of different blots to identify the presence of whichever molecule they are focused on, including RNA, DNA, or protein, when they are testing a complex combination of related molecules. The process of blotting refers to the transfer of macromolecules, including proteins and nucleic acids, from a gel that is placed onto the solid surface of an immobilized membrane so that the specific molecules can be detected. All blotting techniques share a very similar workflow, which is why different methods have to be used to differentiate them from each other. Those methods are referred to as the Northern Blot, the Southern Blot, and the Western Blot methods.
The Western Blot: A little explanation
Western blotting is used to locate specific proteins in a substance, while the Northern Blot is used to determine the size and the number of RNA transcripts from a gene the lab technician is interested in.
Southern and Northern Blotting
Southern blotting was first developed in 1975 by Edwin Southern, hence its name. It was created as an analytical technique used in molecular biology research that allows users to measure the amount and size of very specific DNA sequences that are located in a complex combination through the immobilization of the target sequence to a solid support. It is then sized with a complementary DNA prep. Northern blotting was created not long after the Southern blotting method, so the two could be used in conjunction with each other, since they both share a similar workflow. This workflow includes using a sample preparation of a very purified and high-quality RNA within a substance and utilizing gel electrophoresis to separate the nucleic acid fragments by their sizes before then transferring, or in this case will refer to it as blotting, to a solid support area that will immobilize the isolated nucleic acid that has been targeted. The preparation and hybridization phase then occurs in the nucleic acid probe so that the detection of the nucleic acid probe can be determined.
When lab technicians or students prepare a sample to be used in this experiment, keep in mind that Southern and Northern blots can both begin nearly the same way. The target DNA or RNA is purified from the samples by using nucleic acid extraction. The most common techniques to be used in this process are filter-based, spin basket extraction, and magnetic particle methods.
The next step in both the Northern and Southern Blotting processes is the gel electrophoresis phase. It is used to isolate different proteins and their markers along with RNA and DNA bands. This system separates it out.
After the electrophoresis is done, the DNA or RNA is transferred over to a membrane of nylon that is positively charged. The kit by Modern Biology, Inc. has everything you need to complete the entire process and allows for a much quicker outcome of the process. You will not even need any liquids or buffers, which will make the whole thing come to completion even sooner.
The next step in the process is probe labeling. In this step, a probe is used on the nucleic acid to facilitate the homologous to the sequences that are targeted. They are then labeled with a dye, an enzyme, or through radioactivity which generates a chemical signal to let you know that it is luminescent after incubation.
Next up is the hybridization process in which the labeled probe is incubated with the DNA or RNA fragments that are on the blot so that hybridization can occur. This process within the kit by Modern Biology, Inc. takes less than two hours to complete.
The last step is to go over your results to see if the marker that you were looking for has been detected.
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