nf-κb RelA/p65 protein site-specific phosphorylation diagnostic kit
A technology of p65 and kits, applied in fluorescence/phosphorescence, biological testing, instruments, etc., can solve problems such as less success
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Embodiment 1
[0036] Example 1: Preparation of a sandwich time-resolved fluorescence immunoassay (TRFIA) kit for the detection of NF-κB RelA / p65 Ser276 and Ser536 phosphorylated proteins
[0037] In this embodiment, the sandwich time-resolved fluorescence immunoassay (TRFIA) kit includes:
[0038] Plates or beads coated with specific capture monoclonal antibodies;
[0039] Biotin or other labeled specific detection antibody from another genus;
[0040] Coating buffer (1×PBS, pH7.3±0.1);
[0041] Sample diluent (1×PBS, 1%BSA, 0.05%NaN3, pH 7.3±0.1);
[0042] Blocking solution (1×PBS, 3% BSA);
[0043] Eluent PBST (1×PBS, 0.05% Tween-20);
[0044] Streptavidin-Eu / HRP / ALP;
[0045] The biotin labeling kit was purchased from Pierce Company. Streptavidin-Eu was purchased from Perkin Elmer. Prepare the time-resolved fluorescence immunoassay (TRFIA) plate for detection by yourself, dilute the capture antibody to 7-10 μg / ml with coating buffer, and add 100 μl of the above to each well of the t...
Embodiment 2
[0049] Example 2: Detection of phosphorylation levels of NF-κB RelA / p65 Ser276 and Ser536 in tumor tissues of lung cancer patients by sandwich time-resolved fluorescence immunoassay (TRFIA) kit
[0050] Preparation of detection time-resolved fluorescence immunoassay plate: Dilute the capture antibody to 7-10 μg / ml with coating buffer, add 100 μl of the above antibody dilution to each well of a time-resolved fluorescence immunoassay (TRFIA) plate, overnight at 4ºC ; Take out, put at 37ºC for 30 minutes; shake off the liquid, wash 5 times with PBST; air dry, add 100 μl blocking solution to each well, 37ºC for 2 hours; shake off the liquid, wash 2 times with PBS; air dry naturally at room temperature 2 hours; vacuum seal with foil, set at 4ºC, set aside.
[0051] Detection of tissue samples: The soluble protein solution of tumor tissue was prepared by standard methods, diluted 1:10 with PBS, and 100 μl of sample diluent or standard (different concentrations of RelA / p65 Ser276 or ...
Embodiment 3
[0055] Example 3: Detection of phosphorylation levels of NF-κB RelA / p65 Ser276 and Ser536 in tumor tissues of colon cancer patients by sandwich time-resolved fluorescence immunoassay (TRFIA) kit
[0056] Preparation of detection time-resolved fluorescence immunoassay plate: Dilute the capture antibody to 7-10 μg / ml with coating buffer, add 100 μl of the above antibody dilution to each well of a time-resolved fluorescence immunoassay (TRFIA) plate, overnight at 4ºC ; Take out, put at 37ºC for 30 minutes; shake off the liquid, wash 5 times with PBST; air dry, add 100 μl blocking solution to each well, 37ºC for 2 hours; shake off the liquid, wash 2 times with PBS; air dry naturally at room temperature 2 hours; vacuum seal with foil, set at 4ºC, set aside.
[0057] Detection of tissue samples: The soluble protein solution of tumor tissue was prepared by standard methods, diluted 1:10 with PBS, and 100 μl of sample diluent or standard (different concentrations of RelA / p65 Ser276 or...
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