Document
Generically Dependent Continuant
National Science Foundation, NSF: CA05388, PCM-835 1884
RDF description of Highly sensitive immunoadsorption procedure for detection of low-abundance proteins - http://orbis.uaslp.mx/vivo/individual/scopus193
National Cancer Institute, NCI: P01CA005388, R01CA035547
Article
dodecyl sulfate sodium, 151-21-3; protein, 67254-75-5; staphylococcus protein A, 37380-84-0; Proteins; Sodium Dodecyl Sulfate, 151-21-3; Staphylococcal Protein A; Viral Proteins
critical micelle concentration; fluorography; low-abundance proteins; protein A; sodium dodecyl sulfate; staph A immunoadsorption dodecyl sulfate sodium; protein; staphylococcus protein A; virus protein; protein; article; chemistry; enzyme linked immunosorbent assay; micelle; Mouse mammary tumor oncovirus; precipitation; solubility; temperature; antigen antibody complex; immunoadsorption; methodology; micelle; nonhuman; priority journal; Immunosorbent Techniques; Mammary Tumor Virus, Mouse; Micelles; Microchemistry; Precipitation; Proteins; Sodium Dodecyl Sulfate; Solubility; Staphylococcal Protein A; Support, Non-U.S. Gov't; Support, U.S. Gov't, Non-P.H.S.; Support, U.S. Gov't, P.H.S.; Temperature; Viral Proteins; mammary tumor virus; Mouse mammary tumor virus; tumor virus
Highly sensitive immunoadsorption procedure for detection of low-abundance proteins
A procedure that virtually eliminates nonspecific adsorption of radiolabeled proteins during immunoprecipitation was devised utilizing staphylococcal cells containing protein A (Staph A). Immunoprecipitates (antigen-antibody complexes) were solubilized from Staph A pellets into detergent micelles by incubation in a small volume of 1%25 sodium dodecyl sulfate (SDS) at 23°C for 10 min. To allow re-formation of immunocomplexes and rebinding to new Staph A, the SDS-solubilized material was diluted 20-fold in buffer containing 1%25 Triton X-100 and 0.5%25 sodium deoxycholate. Specific conductance measurements revealed that this solubilization and subsequent reimmunoadsorption of antibody-antigen complexes occur at SDS concentrations that are first above and then below its critical micelle concentration. This procedure lowered the nonspecific background from approximately 2250 parts per million (ppm) to less than 25 ppm with a final recovery of 30-50%25 depending on the antigen and antibody. Chaotropic agents such as 2 m urea, 0.2 m KOH, and 3.5 m MgCl2 (as well as combinations of urea and SDS) can substitute for 1%25 SDS, although the final recovery is somewhat lower. Fluorography of radiolabeled proteins obtained in this manner displays virtually undetectable background even for exposures as long as 2 months. These methods allowed the unambiguous detection of low-abundance antigens at a high level of sensitivity, for example, mouse mammary tumor virus protein products and epidermal growth factor receptor. © 1986.
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