Abstract：
:Protein phosphorylation is a major molecular mechanism by which cellular function is regulated. In order to accomplish rapid and specific biochemical changes via phosphorylation, the activity of a protein kinase must be dynamically regulated. Historically, the activity of each protein kinase has been analyzed using a unique in vitro biochemical assay with a specific substrate and detection procedure. These assays require the use of radioactivity and are often labor intensive. Upon activation, most protein kinases autophosphorylate. Thus, a technical approach to detect changes in kinase activity is to measure autophosphorylation. The purpose of this protocol is to provide a detailed stepwise procedure for measuring the regulation of Src-class kinase activity using phosphorylation state-specific antibodies. Antibodies to a phosphorylated peptide derived from the autophosphorylation site of Src-family kinases are developed and affinity purified. The purified antibodies are used to analyze the regulation of Src and Fyn activity in a mouse muscle cell line. It is anticipated that the utility of these phosphorylation state-specific antibodies will ultimately result in the development of similar antibodies useful for analyzing the activity of many different kinases.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Miermont AM,Mohamed AS,Swope SLdoi
10.1016/s0022-1759(00)00292-1keywords:
subject
Has Abstractpub_date
2000-12-01 00:00:00pages
203-15issue
1-2eissn
0022-1759issn
1872-7905pii
S0022-1759(00)00292-1journal_volume
246pub_type
杂志文章abstract：:Immune complexes attached to and ingested by human polymorphonuclear (PMN) cells were quantified by cytofluorometry using a fluorescence quenching assay which permits differentiation between attachment and ingestion. The fluorescence intensity decreased after ingestion as a result of the low pH in the phagolysosomes. ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(83)90340-x
更新日期：1983-05-27 00:00:00
abstract：:A competition radioimmunoassay for human superoxide dismutase (SOD-1) is described. Radioiodinated SOD-1 is incubated with unlabeled competitor and limiting antibody, then immune complexes are isolated using Staphylococcus aureus. The assay is sensitive (detecting 5 ng enzyme) and reproducible. Average values for SOD-...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(79)90313-2
更新日期：1979-01-01 00:00:00
abstract：:To date, phase I/II dendritic cell (DC)-based cancer vaccine trials have required repeated venesection or leukapheresis to generate the DCs. Previous studies have suggested that DCs may be cryopreserved and revived for clinical use as sequential immunisations. We have developed a method of cryopreserving monocyte-deri...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(02)00430-1
更新日期：2003-01-15 00:00:00
abstract：:The detection of site-directed anti-idiotypic antibodies is usually based on their ability to inhibit the binding of antigen to idiotype, in either solid- or fluid-phase radioimmunoassays. Passage of serum over antigen-coupled immunosorbents for the purpose of removing the idiotypes from complexes with putative anti-i...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(87)90172-4
更新日期：1987-06-26 00:00:00
abstract：:We describe a simple, rapid and reproducible in vitro culture system in which human peripheral blood lymphocytes (PBLs), donated 24 h prior to initiation of culture can be stimulated to produce antigen-specific antibodies. Peripheral blood lymphocytes purified by Ficoll-Hypaque centrifugation were passed over a G10 Se...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(95)00256-1
更新日期：1996-03-28 00:00:00
abstract：:A system proposed for measurement of mutational risk consists in detection of hemoglobin mutations expressed in erythrocytes. For this detection the production of antibodies specific for Hb variants is essential. Recently we reported a sensitive solid-phase EIA for the production and selection of polyclonal and monocl...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(83)90189-8
更新日期：1983-05-13 00:00:00
abstract：:Direct reverse transcriptase in situ polymerase chain reaction (RT-in situ PCR) of selected mRNA expression in rat mast cells (MC) and alveolar macrophages (AM) was optimized. Rat peritoneal mast cells (PMC), rat cultured mast cells (RCMC), rat bronchoalveolar lavage cells (BALC) or rat cultured alveolar macrophages (...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(99)00061-7
更新日期：1999-06-24 00:00:00
abstract：:We have developed a modified microtiter plate which has following advantageous features and functions to both conventional microtiter plate and protein array, such as 1) use of conventional microtiter plate reader and washer, and 2) allowance of simultaneous reaction in the same liquid for all wells. Four proteins of ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2007.04.007
更新日期：2007-06-30 00:00:00
abstract：:A fast and simple indirect immunoperoxidase staining technique is described, which can also be used for the characterization of cells with high endogenous peroxidase activity such as many haemopoietic cells. It is based on the combination of a newly developed glucose-oxidase plus glucose procedure for the inhibition o...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(86)90267-x
更新日期：1986-01-22 00:00:00
abstract：:An anti-cocaine monoclonal antibody (mAb) designated h2E2 will soon enter clinical trials for the treatment of cocaine abuse disorders. Importantly, this antibody selectively binds cocaine and its active metabolite, cocaethylene, with high affinity, while binding inactive metabolites with substantially lower affinitie...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2019.112676
更新日期：2020-01-01 00:00:00
abstract：:This article describes a novel method for detecting anti-endothelial cell antibodies (AECAs). Sera from patients with systemic vasculitis or inflammatory conditions have been reported to contain antibodies (Abs) that bind to endothelial cells (EC), i.e., AECAs. AECAs are known to play immunogenic effects by triggering...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2012.05.002
更新日期：2012-08-31 00:00:00
abstract：:The guinea pig has a storied history as a model in the study of infectious disease and immunology. Because of reproducibility of data and availability of various reagents, inbred mice have since supplanted the guinea pig as the animal model-of-choice in these fields. However, several clinically-significant microorgani...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2013.01.005
更新日期：2013-03-29 00:00:00
abstract：:A simple hemolytic plaque assay for the detection of red blood cells carrying abnormal or mutant hemoglobin is described. The assay is based on the use, as indicator cells, of sheep red blood cells coated with monospecific antibody against the hemoglobin variant, another complement fixing multispecific anti-hemoglobin...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(82)90075-8
更新日期：1982-11-26 00:00:00
abstract：:A high-cloning efficiency, filler cell-free limit-dilution culture system for the growth and differentiation of single cytotoxic T lymphocyte precursors (CTLp) was tested for its ability to maintain the lytic specificity of the resultant clones of cytotoxic T lymphocytes (CTL). The system used non-specific stimulation...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(91)90049-l
更新日期：1991-10-25 00:00:00
abstract：:IL-2 activity is commonly estimated in cell culture supernatants by an IL-2-dependent cell proliferation assay. This method is both reproducible and sensitive. However, it often appears from the literature that statistical analysis of the titration data either is disregarded or, when performed, is based on statistical...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(86)90463-1
更新日期：1986-02-12 00:00:00
abstract：:Hybridomas secreting monoclonal antibodies specific for hemoglobin nonenzymatically glycated in the non-A1c position were produced by fusion of SP 2/0 myeloma cells with spleen cells from BALB/c mice immunized with nonenzymatically glycated hemoglobin prepared from human erythrocytes. Wells containing hydridomas secre...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(91)90364-l
更新日期：1991-07-05 00:00:00
abstract：:A new and simple method for quantitation of IgM of low molecular weight in sera by radial immunodiffusion in 7% agar gel is described. The test is easy to perform and permits exact determination of low molecular weight IgM concentration down to very small quantities (1 mg%). Results obtained with the method described ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(78)90094-7
更新日期：1978-01-01 00:00:00
abstract：:Small haptens such as methylamphetamine (MW 149) cannot, on their own, induce an immune response. It is also unlikely that they fill the binding site of any antibody that recognises them. Under such circumstances any attached label might be expected to enter the area of the binding site and exert an influence on overa...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(91)90375-p
更新日期：1991-07-05 00:00:00
abstract：:The oxidative burst generation capacity of granulocytes can be reliably detected by a flow cytometric procedure using lysed whole blood, dihydrorhodamine 123 (DHR), and phorbol myristate acetate (PMA). This assay is used to detect chronic granulomatous disease (CGD) and the CGD carrier state. To assess the feasibility...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(95)00210-3
更新日期：1995-12-15 00:00:00
abstract：:Human peripheral blood lymphocytes were stained simultaneously with the lipophilic membrane label PKH-2 and surface markers and analysed by multiparameter flow cytometry. A biphasic staining distribution was observed for T (CD3+) as well as B (CD19+) cells. Within the T subset, CD4+ cells stained almost exclusively wi...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(92)90211-b
更新日期：1992-09-18 00:00:00
abstract：:A paper radioimmunosorbent test (PRIST) was shown to be sensitive and reproducible when used with excretory/secretory antigen of Toxocara canis second stage larvae. Whatman No. 50 filter paper (5 mm discs) gave the most consistent and clear results with antigen at a concentration of 100 micrograms/ml, and could be sto...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(80)90180-5
更新日期：1980-01-01 00:00:00
abstract：:A competitive radioimmunoassay for immunoconglutinin (IK) was developed using isolated antostimulated rabbit IK labelled with 125I and glutaraldehyde-fixed sheep erythrocytes coated with fixed complement (EAC). IKs in test sera of human or animal origin produced specific inhibition of uptake of [125I]IK onto EAC and t...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(80)90045-9
更新日期：1980-01-01 00:00:00
abstract：:To date, the applications of bispecific antibodies in immunoassay and immunocytochemical procedures have been directed at uniting two different biomolecules through the binding of epitopes on each respective substance. In this study, bispecific antibodies were constructed in which both binding sites were directed to t...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(94)90042-6
更新日期：1994-05-16 00:00:00
abstract：:We have developed a sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of human heart type fatty acid-binding protein (H-FABP) in human plasma and urine using the combination of two distinct monoclonal antibodies (MAbs) directed against human H-FABP purified from human heart muscle. The total ass...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(94)00248-u
更新日期：1995-01-13 00:00:00
abstract：:A sensitive, simple and reproducible biotin-avidin amplified double antibody immunoassay to quantitate low concentrations of mouse immunoglobulins is described. The assay is a useful technique to measure trace levels of murine monoclonal antibodies in culture supernatants of hybridoma cells metabolically labeled with ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(85)90355-2
更新日期：1985-10-10 00:00:00
abstract：:Human phospholipase B-precursor (PLB-P) is a newly identified and purified protein from human neutrophils. The precise function of PLB-P in vivo is not yet known. Its existence in neutrophils and the enzymatic activity against phospholipids imply a role in the defence against invading microorganisms and in the generat...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2010.01.005
更新日期：2010-02-28 00:00:00
abstract：:Many normal and pathological processes have been shown to be influenced by the quantitative and qualitative nature of cytokine production. Cytokines have also been shown to be central in modulating host responses to invasion by pathogens. Therefore detection and analysis of cytokine production are critical in the unde...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/s0022-1759(97)00124-5
更新日期：1998-03-01 00:00:00
abstract：:Paraneoplastic neurological syndromes (PNS) are devastating neurological disorders secondary to cancer, associated with onconeural autoantibodies. Such antibodies are directed against neuronal antigens aberrantly expressed by the tumor. The detection of onconeural antibodies in a patient is extremely important in diag...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2013.02.017
更新日期：2013-05-31 00:00:00
abstract：:A simple and rapid method was developed for immunofluorescence measurements of cells by flow cytometry which does not require washing procedures, permitting absolute enumeration of cell subpopulations. Peripheral blood cells were labeled with fluorescein and phycoerythrin conjugated monoclonal antibodies and the nucle...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/0022-1759(89)90034-3
更新日期：1989-09-29 00:00:00
abstract：:CD4(+) T cells play a central role in regulating the immune response. Their effector function is commonly assessed by their capacity to secrete cytokines detected by ELISPOT and intracellular cytokine staining. However, one aspect of their effector function that is often overlooked is their ability to help activation ...
journal_title：Journal of immunological methods
pub_type： 杂志文章
doi：10.1016/j.jim.2012.10.013
更新日期：2013-01-31 00:00:00
