Blotting techniques

Most sample components analyzed with electrophoretic techniques are invisible to the naked eye. Thus methods have been developed to visualize and quantify separated compounds. These techniques most commonly involve chemically fixing and then staining the compounds in the gel. Other detection techniques can sometimes yield more information, such as detection using antibodies to specific compounds, which gives positive identification of a sample component either by immunoelectrophoretic or blotting techniques, or enhanced detection by combining two different electrophoresis methods in two-dimensional electrophoretic techniques. 

The northern blotting technique is similar to the Southern blotting technique with the exception that northern blots detect specific sequences of RNA, not DNA. 

Blotting techniques may be used in a variety and combination of electrophoretic systems which makes their use widespread and convenient when protein concentrations are minimal and agarose or polyacrylamide is the matrix choice. 

Transfer (blot) gel to nitrocellulose filter using Soudiern blot technique... 

Southern blotting Technique used to identify and locate DNA sequences which are complementary to another piece of DNA called probe using electrophorectic gels for separation of DNA and membrane filters with radiolabelled complementary probes. 

Third Step The Western blotting technique, applied to cell extracts, was used to confirm the pRB immunostaining results in two bladder cancer cell lines of T24 and J82, giving quantitative results for pRB in the two cell lines, comparable with that demonstrated by IHC (Fig. 1.3). 

Diamandis, E.P. (1993) Time-resolved fluorometry in nucleic acid hybridization and Western blotting techniques (Review). Electrophoresis 14, 866-875. 

BLOTTING is a method to detect specific DNA (or RNA) fragments that contain sequences that are complementary to sequences in the labeled probe molecule. Only a few of the many DNA fragments on a gel will contain the sequence of interest, and only these will be seen (light up) on the blot. Specific proteins can also be visualized by blotting techniques using a specific antibody to detect a specific protein. 

These blotting techniques are known by the names of compass directions (Southern, Northern, Western). Since Southern is a person s name, there s no logic in how the different blots were named. Southern developed a blot in which DNA on the blot is detected by a labeled DNA probe. It was then fairly logical that the next technique developed, detecting RNA on the blot with a DNA probe, should be called a Northern blot. Then things got carried away with the Western, and now the Southwestern, and so on and so on. 

West Basin Reclamation Plant, 26 84 Western blotting technique, 9 756... 

Genes cloned as cDNA are used for gene therapy protocols and for producing transgenic animals. cDNA probes, for many of the blotting techniques, are produced by doning. 

Patients and blood donors are routinely screened for exposure to HIV by means of ElISA and Western blot assays of blood samples (F uie 1-7-15). The assays are designed to detect antibodies to HIV in the blood of the test subject The ELISA is used as the primary screening assay because it is very sensitive. Because the reference interval for the test is set to include everyone with antibodies to HIV, it also gives false positives and thus has a rather low positive predictive value, especially in low-risk populations. The Western blot (or immunoblot) is used as the confirmatory test for HIV exposure. In the Western blot technique, specific HIV proteins are separated by gel electrophoresis and blotted to a filter. The filter is incubated with the test sample. If the sample contains antibodies to HIV, they will bind to the proteins on the filter. The filter is next washed and incubated with a labeled goat anti-human IgG to visualize any bound human antibodies. The Western blot is highly specific. The combination of an ELISA and Western blot has a positive predictive value of greater than 99%,... 

Transfer by blotting to a nylon membrane allows the heat treatment involved in hybridisation to take place. Southern blotting technique. 

Figure 3.24 Restriction fragments from digestion of two samples of DNA. These are produced as a result of the activity of restriction enzymes on the samples of DNA and separation of the fragments by the Southern blotting technique (Appendix 3.12). Numbers refer to the length of the fragments, (i.e. number of nucleotides).

---