2-dimensional SDS-PAGE uses the principles and techniques outlined above. 2-D SDS-PAGE, as the name suggests, involves the migration of polypeptides in 2 dimensions. For example, in the first dimension polypeptides are separated according to isoelectric point, while in the second dimension polypeptides are separated according to their molecular weight. The isoelectric point of a given protein is determined by the relative number of positively (e.g. lysine and arginine) and negatively (e.g. glutamate and aspartate) charged amino acids, with negatively charged amino acids contributing to a high isoelectric point and positively charged amino acids contributing to a low isoelectric point. Samples could also be separated first under nonreducing conditions using SDS-PAGE and under reducing conditions in the second dimension, which breaks apart disulfide bonds that hold subunits together. SDS-PAGE might also be coupled with urea-PAGE for a 2-dimensional gel.
In principle, this method allows for the separation of all cellular proteins on a single large gel. A major advantage of this method is that it often distinguishes between different isoforms of a particular protein - e.g. a protein that has been phosphorylated (by addition of a negatively charged group). Proteins that have been separated can be cut out of the gel and then analysed by mass spectrometry, which identifies the protein.
Please refer to reference articles for examples of the application of 2-D SDS PAGE.
Medical diagnostic applications
The confirmatory HIV test employs a Western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane as above. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added. The stained bands then indicate the proteins to which the patient's serum contains antibody.
A Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease').
Some forms of Lyme disease testing employ Western blotting.
Western blot can also be used as a confirmatory test for Hepatitis B infection.
In veterinary medicine, Western blot is sometimes used to confirm FIV+ status in cats.
. When examining foods, the possibility of detecting the presence of microorganisms by looking at a sample directly under the microscope should not be missed.
. A small amount of material can be mounted and teased out in a drop of water on a slide, covered with a cover slip, and examined.
Direct-to-Consumer genetic testing
Direct-to-Consumer (DTC) genetic testing is a type of genetic test that is accessible directly to the consumer without having to go through a health care professional. Usually, to obtain a genetic test, health care professionals such as doctors acquire the permission of the patient and order the desired test. DTC genetic tests, however, allow consumers to bypass this process and order one themselves. There are a variety of DTC tests, ranging from testing for breast cancer alleles to mutations linked to cystic fibrosis. Benefits of DTC testing are the accessibility of tests to consumers, promotion of proactive healthcare and the privacy of genetic information. Possible additional risks of DTC testing are the lack of governmental regulation and the potential misinterpretation of genetic information.
3.Explain the culture techniques in detail.
The full microbiological examination usually requires that individual viable propagules are encouraged to multiply in liquid media or on the surface, or with in the matrix, of a medium solidified with agar.
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Epithelial cellsin culture, stained for keratin (red) and DNA (green)
Cell culture is the complex process by which cells are grown under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes, including viruses, bacteria and protists. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture.
Animal cell culture became a common laboratory technique in the mid-1900s, but the concept of maintaining live cell lines separated from their original tissue source was discovered in the 19th century.