Optimization of immunochemical methods for intracellular protein detection.
Given the possibility that cell kinetics and p53 status may be important determinants of chemotherapeutical efficacy, the aim of the present study was to determine the optimal methods and conditions for qualitative and quantitative intracellular proteins detection. Bradford assay is the better choice for protein concentration detection because it is more sensitive and more rapid than Sheffield assay despite the fact that it utilizes a higher amount of samples. The direct staining method for flow-cytometrical detection of intracellular proteins is more rapid as compared to the indirect staining one, also providing information about protein expression during cell cycle phases, but it is low sensitive for protein expression estimation and is prohibitive for masked intracellular proteins like PCNA. More than that, it can be performed with both fixed and freshly isolated cells as compared to the indirect staining method, but the last one provides advantages by signal amplification and by its availability of using it for a large number of intracellular proteins detection.
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St. S. Nicolau Institute of Virology, Center of Immunology, Bucharest.
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D005434:Flow Cytometry / Q000379:methods / Q000592:standards; D019084:Fluorescent Antibody Technique, Indirect / Q000592:standards; D018922:HL-60 Cells; D006801:Humans; D015151:Immunoblotting / Q000379:methods / Q000592:standards; D007120:Immunochemistry / Q000379:methods / Q000592:standards; D007424:Intracellular Fluid / Q000737:chemistry; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D016159:Tumor Suppressor Protein p53 / Q000032:analysis; D020298:U937 Cells
D018809:Proliferating Cell Nuclear Antigen; D011506:Proteins; D016159:Tumor Suppressor Protein p53