Figure 1. Target Map of the PathScan® Cancer Phenotype Antibody Array Kit (Fluorescent Readout) #14822.
Figure 2. HeLa and A-431 cells (Panel A), and HCC827 and Calu-3 cells (Panel B) were grown to 90% confluency. Cell Lysates were prepared and analyzed using PathScan® Cancer Phenotype Antibody Array Kit (Fluorescent Readout) #14822. Array fluorescent images were acquired using the LI-COR® Biosciences Odyssey® Imaging System (upper) and fluorescence intensities for each spot were quantified using the LI-COR® Image Studio v2.0 array analysis software and depicted as bar graphs (lower).
Figure 3. A-431 cells were treated for five days with a cocktail of EMT inducers. Cell lysates were prepared and analyzed using the PathScan® Cancer Phenotype Antibody Array Kit (Fluorescent Readout) #14822. Array fluorescent images were acquired using the LI-COR® Biosciences Odyssey® imaging system (upper) and fluorescence intensities for each spot were quantified using the LI-COR® Image Studio v2.0 array analysis software and depicted as a bar graph (lower).
Figure 4. Various cells (indicated) were grown and cell extracts were prepared and analyzed using the PathScan® Cancer Phenotype Antibody Array Kit (Fluorescent Readout) #14822. Slides were scanned using the LI-COR® Biosciences Odyssey CLx infrared imaging system. Fluorescence intensity for each spot was quantified using the LI-COR® Biosciences Image Studio v2.0 array analysis software. Duplicate spot intensities were averaged and values were normalized after subtracting the background signal. The heatmap was generated using MultiExperiment Viewer (MeV) analysis software.
The PathScan® Cancer Phenotype Antibody Array Kit (Fluorescent Readout) uses glass slides as the planar surface and is based upon the sandwich immunoassay principle. The array kit allows for the simultaneous detection of 19 cancer cell associated proteins. Target-specific capture antibodies have been spotted in duplicate onto nitrocellulose-coated glass slides. Each kit contains two slides allowing for the interrogation of 32 different samples and the generation of 608 data points in a single experiment. Cell lysates are incubated on the slide followed by a biotinylated detection antibody cocktail. DyLight™ 680-linked Streptavidin is then used to visualize the bound detection antibody. A fluorescent image of the slide can then be captured with a digital imaging system and spot intensities quantified using array analysis software.
PathScan® Cancer Phenotype Antibody Array Kit (Fluorescent Readout) detects the target proteins as specified on the Array Target Map. No substantial cross-reactivity has been observed between targets. This kit is optimized for cell lysates diluted to a total protein concentration between 0.2 and 1 mg/ml (see kit protocol). All sandwich assays have been validated for human derived samples. This kit detects proteins from the indicated species as determined through in-house testing, but may also detect homologous proteins from other species.
Despite shared hallmarks and common overarching principles, cancers are heterogeneous in nature. Widely used tumor derived cell lines often exhibit both genomic and proteomic differences relative to one another. Factors contributing to these differences include gradual accumulation of genetic lesions within cell lines, transcriptional networks and epigenetic marks remaining from the original tissue, and various adaptations to in vitro culture conditions. Cell lines may differ considerably in the architectural details of basic machineries, the wiring of signal transduction circuitry, and the set points of metabolic pathways. As a result, heterogeneous phenotypes within cell lines will manifest as differences in fundamental cellular functions and a range of behaviors under experimental conditions.
The cell surface represents a compartment where a high level of heterogeneity is displayed. Cancer cells can use the wide array of cell surface proteins to sense, attract, and respond to survival or growth factors in order to promote cancer cell proliferation and growth. Certain cell surface molecules can shield cancer cells from the immune response, help to procure nutrients and create more favorable conditions, and provide a means for the spread and colonization of the cancer cells to other tissues. The presence (or absence) of specific cellular proteins can be regarded as a characteristic phenotypic trait that allows for the classification of cancer cell type and may help predict cellular response to specific experimental conditions both in vitro and in vivo.
Understanding and modeling tumor cell behavior, sensitivity and resistance to various drugs, identification of oncogene dependencies and network vulnerabilities requires a systems-level measurement of multiple parameters. Therefore, it is important to simultaneously survey both the cell surface as well as key molecules that serve as indicators for cell cycle progression, epithelial to mesenchymal transition, and activation of the hypoxic response program.
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.
LI-COR is a registered trademark of LI-COR, Inc.
Odyssey is a registered trademark of LI-COR, Inc.