C5F2-HCB

Kinase activity assays measure the ability of kinases to transfer phosphate groups from ATP to specific substrates, while phosphorylation assays detect the presence and levels of phosphorylated proteins. Common methods include radiolabeled ATP incorporation (e. g. ,), ADP release detection via bioluminescence (e. g. ,[3]), enzyme-linked immunosorbent assays (ELISA) for phospho-specific epitopes (e. g. ,[6]), and microtiter-based formats for high-throughput screening (e. g. ,[8]). The ADP-Glo assay quantifies kinase activity by measuring ADP produced during phosphorylation using a luciferase-based system. Radiometric assays involve autoradiography or scintillation counting after incorporation of ³²P-labeled ATP into substrate proteins. ELISA-based approaches rely on phospho-specific antibodies to detect activated kinases in cell lysates or purified samples.

Western blotting (WB) is a commonly used experimental method in molecular biology, biochemistry, and immunogenetics for identifying and quantifying target proteins. It combines gel electrophoresis with immunoassay, enabling researchers to analyze protein expression, post-translational modifications, and molecular weight.

Multiplex immunofluorescence IHC detects multiple protein biomarkers in one tissue section by sequential antibody staining, HRP-mediated tyramide fluorophore deposition, heat-mediated antibody stripping, nuclear counterstaining, multispectral imaging, spectral unmixing, and digital cell phenotyping; TSA deposits fluorophore near the antigen so the fluorescence signal remains after primary and secondary antibodies are removed, enabling repeated staining cycles, including with antibodies from the same host species. Classic FFPE tumor immune-profiling applications use panels such as CD3, CD8, CD68/CD163, FOXP3, PD-1, PD-L1, pancytokeratin, Ki67, and DAPI to identify tumor cells, immune-cell subsets, checkpoint-marker expression, co-expression phenotypes, cell density, and spatial relationships in the tumor microenvironment.

Cytotoxicity assays are usually based on the assessment of cell membrane damage, which can also be indirectly detected by measuring cell viability. Detection methods include MTT assay, CKK-8 assay, LDH assay and ATP assay, etc.

Multiplex immunohistochemistry (mIHC), also known as tyramide dignal amplification (TSA), is an enzymatic detection method that uses horseradish peroxidase (HRP) to perform high-density in-situ labeling of target proteins or nucleic acids.

Kinase activity assays measure transfer of phosphate from ATP to a protein or peptide substrate, generating phosphorylated substrate, ADP, or incorporated radiolabeled phosphate as the readout; phosphorylation assays measure site-specific phosphorylation in cells or tissues as a proxy for kinase-pathway activation, inhibition, or substrate regulation. Phosphorylation can be detected by phospho-specific Western blot, immunoprecipitation kinase assay, phospho-immunofluorescence, phospho-flow cytometry, luminescent ADP detection, radiolabeled ATP incorporation, or reporter-based pathway assays, and these readouts can be applied to cancer cells, primary neurons, mouse tumors, organoids, inflammatory macrophages, ferroptosis studies, and mitophagy studies when the kinase target is biologically relevant.

Breast cancer is a heterogeneous cancer, and it has been distinguished into four subtypes: luminal A, luminal B, HER2-positive and basal-like. Molecular mutations, epigenetic alterations, hormone exposure and immune microenvironment are related to the progression of breast cancer.

Cancer immunology studies how the immune system recognizes, suppresses, edits, or fails to eliminate malignant cells through tumor antigen release, antigen presentation, T-cell priming, immune trafficking, tumor-cell killing, and feedback inhibition in the tumor microenvironment. The cancer-immunity cycle links tumor antigenicity, dendritic-cell priming, CD8+ T-cell infiltration, cytotoxic function, and immune-checkpoint regulation to tumor rejection or immune escape. Immune-checkpoint pathways such as PD-1/PD-L1 and CTLA-4 suppress antitumor T-cell activity and can be therapeutically blocked, but many tumors remain resistant because of poor antigen presentation, weak T-cell infiltration, suppressive myeloid cells, regulatory T cells, and tumor-intrinsic immune-exclusion programs. Unresolved questions include which immune-cell states predict response, how tumor-intrinsic pathways exclude immune cells, how myeloid suppression limits checkpoint blockade, and which combination strategies