By Ronald B. Corley
Millions of equipment were constructed within the a number of biomedical disciplines, and people lined during this booklet characterize the elemental, crucial and most generally used tools in different various disciplines.
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Extra resources for A Guide to Methods in the Biomedical Sciences
It is therefore analogous to Southern blotting for DNA. The only major difference is that because RNA can form secondary structures, it must be resolved on gels under denaturing conditions. Common denaturants include formaldehyde and glyoxal. Northern blotting got its name from its developers, Alwine and Stark (17), who called it “Northern” blotting as a joke (after “Southern” blotting), but the name has stuck, and other blotting methods have since used compass directions as descriptions as well.
Secondary antibodies are “antibodies against antibodies” that have been labeled with or an enzyme. The use of secondary reagents negates the need to label primary antibodies. In this way, only a small number of secondary antibodies need to be labeled, rather than all of the primary antibodies of interest. Alternatively, biotinylated primary antibodies can be used. Biotin is a small molecular weight vitamin that is strongly bound by avidin, a protein from egg, which can be used as a secondary reagent rather than another antibody.
An electric current is passed at right angels to the gel, and the proteins are transferred onto the membrane. The proteins that have been transferred to the membrane are then detected by a probe, an agent that can selectively and specifically identify a particular protein or related proteins. For western blot analysis, antibodies are the most common probes. The antibodies are directly labeled with a radioactive molecule, such as so that its binding is detectable by autoradiography. Alternatively, it can be labeled with an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP).