Discussion of probing a blot is typical of the kind of comprehension challenge the MCAT writers might put before you on the exam, checking to see that you have basic familiarity with molecular biology technique. Remember, if the discussion in a passage feels high level, don't let the fear take hold. The questions will often be more basic. The question is what kind of feel you have for things, not encyclopaedic knowledge. They do not expect graduate level fluency in molecular biology.

Blotting is a very important technique in molecular biology. Let's keep it simple. In blotting. samples are been transfered onto a membrane, a solid support, by which we mean a sheet of nitrocellulose (fancy paper) or PVDF (polyvinyldifluoride). Usually the transfer is accomplished by applying voltage across a sandwich of the membrane and an electrophoresis gel. The samples (bands) move over from the gel onto the membrane where they get stuck depending on the characteristics of the membrane.

The next stage is '’probing the blot'’. What this means, basically, is that the researcher washes the blot with solutions containing some form molecule (the probe) that will specifically bind to the biomolecules they are interested on the blot. The probes have some characteristic, an attached group or a radiolabel, that will let the researcher detect them later. In the world of nucleic acid blotting (Southerns and Northerns), typically the probe consists of DNA or RNA that is homologous to the sample of interest. It will base pair, get stuck, and be detected. As mentioned, the probe has been modified in some way to produce a detectable signal. With DNA and RNA, usually this is accomplished with 32P labeling, although fluorescent probes are also very common.

In the world of protein blotting (Westerns and ELISA), modified IgG antibodies are typically used to locate and quantitate protein of interest. An antibody will have been raised against the protein of interest (usually by injecting animals) and modified to be detectable. 'monoclonal' and 'polyclonal' are terms of nomenclature you may run into, describing antibodies, usually just to throw you off. If the antibodies are products of many antibody producing cells, they are polyclonal. If the antibodies are the products of a cell population derived from a single cell, they are monoclonal. ELISA (enzyme-linked immunosorbant assay) and Western blotting are two types of immunoassays in wide application. The 'enzyme-linked', 'EL' part of ELISA simply refers to the method of modifying the antibody for detection purposes (analogous to radiolabeling). Certain enzymes, such as horseradish peroxidase, are often bound to antibody probes so that the probes can produce a signal in the detection phase, specifically locating the proteins of interest on the blot. Horseradish peroxidase is used in luminol based systems to produce bursts of light where the samples are located which is recorded photographically.

Blotting is simple. The sample is stuck onto a support membrane and then probed for detection.












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