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Write 7 pages thesis on the topic immunoassay techniques: fluorescence resonance energy transfer and western blotting.
Write 7 pages thesis on the topic immunoassay techniques: fluorescence resonance energy transfer and western blotting. This can be done with an enzyme (enzyme immunoassay EIA), radioisotopes such as I-125 Radioimmunoassay (RIA), or fluorescence. Another method is the Western Blot. Immunoassays are homogeneous or heterogeneous and can be competitive and noncompetitive. "In the competitive immunoassay, the antigen in the unknown sample competes with labeled antigen to bind with antibodies. The amount of labeled antigen bound to the antibody site is then measured. In this method, the response will be inversely proportional to the concentration of antigen in the unknown. This is because the greater the response, the less antigen in the unknown was available to compete with the labeled antigen."(Wikepedia, Immunoassay)Noncompetitive immunoassays are called "sandwich assay." In this method the antigen in the unknown is bound to the antibody site and the labeled antibody is bound to the antigen. A measurement of the labeled antibody is taken. The results will be directly proportional to the concentration of antigen. In heterogeneous assays the unbound antibody must be removed with a reagent, but this step is not necessary with the homogeneous and thus is more convenient and quicker to use.
The transfer of proteins to gel membranes is to provide easier access with probes of which the most common is the antibody. "The power of the technique lies in the simultaneous detection of a specific protein by means of is antigenicity and its molecular mass." (Rybicki 1996)
For the enzyme-assisted immunoelectroblotting (IEB) or Western blot the following equipment is typical.
A vertical slab gel apparatus
Nitrocellulose paper and filter paper, blotting paper cut to size
Blotting electrodes
Blotting buffer -20% methanol and pH above 7.0
Nappy liners-to minimize surface area exposed to electrode
The protein transfer is accomplished by dipping the polyacrylamide gels into the transfer buffer, which is then laid onto nitrocellulose paper that is pre-wetted placed on three layers of wet filter paper that rests on the Anode (+ve electrode). The gel is then overlaid with three wet filter papers and the Cathode (-ve electrode). It is imperative to make sure there are no bubbles between the layers.
Next the completed package is placed on a plastic tray, anode side down. The Anode and Cathode are connected to a power pack with current of 500mA applied for approximately thirty minutes for transfer to occur. The nitrocellulose can be stained with Amido Black, Coomassie Brilliant Blue or Ponceau S staining.
When using the IgG immunoglobulin proteins in Western Blotting electrophoretically separated standard proteins are used to estimate the size of the IgG protein. The IgG protein is multimetric with two heavy chains and two light chains. The heavy chains are indicated by 5 very dark markings arranged horizontally across next to the 50 kilodalton (kDa) standard protein, so the weight of the IgG protein is assumed to be 50 kilodaltons. Further down the nitrocellose membrane are five more markings much lighter near the 25 kilodalton area which are the light chains. The total of the two chains provides the molecular weight of the IgG protein, 150 kilodaltons.
