Antibody insoluble protein preparation

Immunolocalization is a technique for identifying the presence of a protein within the cell, its relative abundance and its subcellular localization. After suitable preparation of the cells, they are treated with an antibody (the primary antibody) that binds to the protein of interest. An antibody that binds to the primary antibody (the secondary antibody) is then allowed to bind and form an antigen—primary antibody—secondary antibody complex. The detection system generally consists of the formation of a colored insoluble product of an enzymatic reaction, the enzyme, such as alkaline phosphatase or horseradish peroxidase, being covalently linked to the secondary antibody. 

Preparations are incubated with appropriate reagents to allow visualization based upon the detection system associated with the secondary antibody. The secondary antibody may be conjugated to a enzyme (e.g., alkaline phosphatase, horseradish peroxidase). Incubation with the appropriate substrate to the enzyme will result in the production of an insoluble colored product that can be detected upon microscopic analyses of the cells. Secondary antibodies can also be conjugated to fluorochromes (e.g., fluorescein, rhodamine) that can be detected using a microscope equipped to detect fluorescence. Immunohisto-chemistry has proven to be a powerful tool in biochemical toxicology allowing for in situ assessments of protein responses to toxicant exposure. 

Reports of examples of arylphosphonates include those of water-soluble phosphinic-polyphosphonic acids, e.g. 132, and the phosphonate 133 which when coupled to alcohols, to give e.g. 134, act as linkers to proteins in experiments intended to generate antibodies to catalyse cationic cyclisation reactions.Novel water-soluble phosphonate-substituted phthalocyanines have been prepared.The phosphonate esters 135 are insoluble in water but can be hydrolysed by hydrochloric acid to give the water-soluble phosphonic acids 136. Aromatic phosphonate-phosphines 137, and their air-stable complexes, have been obtained from the reaction of 4-halogeno-substituted phenylphosphonates with lithium diphenylphosphide. ... 

Immunoquantitation of poIy(ADP-ribose) polymerase from TCA insoluble fractions. Free and hemocyanin-bound poly(ADP-ribose) polymerase from pig thymus (4) was used to prepare polyclonal antibodies in rabbits. The highest titer was obtained with hemocyanin adducts. With the aid of these antibodies, we have developed a procedure for inununoquantitation of poly(ADP-ribose) polymerase. Amounts of antigen could be determined when the TCA precipitates from cells or tissues were dissolved and applied to nitrocellulose. Antibodies specifically binding to the absorbed antigen were then determined with ( I) protein A. The procedure allowed quantitation of polymerase in the ng range (Fig. 1). Determination of cellular contents required only 10 cells. Recovery of added polymerase was almost quantitative. 

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