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Her2 clone 3b5

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HER2 (clone 3B5) is a mouse monoclonal antibody that recognizes the human epidermal growth factor receptor 2 (HER2) protein. HER2 is a member of the EGFR/ErbB family of receptor tyrosine kinases and plays a role in cell growth and differentiation.

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Lab products found in correlation

Duolink pla kit, by Merck Group (2 mentions) Her3 clone d22c5, by Cell Signaling Technology (2 mentions)

2 protocols using her2 clone 3b5

1

Quantifying HER2-HER3 Heterodimer Formation

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We performed PLA to detect the interactions between the c-terminal domains of HER2 and HER3 with the Duolink PLA kit (Sigma-Aldrich) according to the manufacturer’s recommendations with at least 2 biological replicates per sample, and 3 technical replicates. Cells were exposed to growth factors and drug combinations as previously described, then fixed in 4% PFA, permeabilized with Triton X-100, and the Duolink PLA protocol was followed using HER2 (clone 3B5) and HER3 (clone D22C5) antibodies purchased from Cell Signaling Technology. Because of the abundance of HER2-HER3 heterodimers in SKBR3 cells, the assay was slightly modified to reduce detection of total HER2-HER3 dimers for the purpose of more accurate quantification. HER2-HER3 heterodimers were detected as single fluorescent dots in z-series of cells imaged with confocal microscopy. Additionally, cell nuclei were fluorescently stained with DAPI, and cellular cytoskeletons were labeled with tubulin antibody staining. The image analysis software CellProfiler (Kamentsky et al., 2011 (link)) was used to quantify the PLA signal.
Watson S.S., Dane M., Chin K., Tatarova Z., Liu M., Liby T., Thompson W., Smith R., Nederlof M., Bucher E., Kilburn D., Whitman M., Sudar D., Mills G.B., Heiser L.M., Jonas O., Gray J.W, & Korkola J.E. (2018). Microenvironment-Mediated Mechanisms of Resistance to HER2 Inhibitors Differ between HER2+ Breast Cancer Subtypes. Cell systems, 6(3), 329-342.e6.
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2

Quantifying HER2-HER3 Heterodimer Formation

Check if the same lab product or an alternative is used in the 5 most similar protocols
We performed PLA to detect the interactions between the c-terminal domains of HER2 and HER3 with the Duolink PLA kit (Sigma-Aldrich) according to the manufacturer’s recommendations with at least 2 biological replicates per sample, and 3 technical replicates. Cells were exposed to growth factors and drug combinations as previously described, then fixed in 4% PFA, permeabilized with Triton X-100, and the Duolink PLA protocol was followed using HER2 (clone 3B5) and HER3 (clone D22C5) antibodies purchased from Cell Signaling Technology. Because of the abundance of HER2-HER3 heterodimers in SKBR3 cells, the assay was slightly modified to reduce detection of total HER2-HER3 dimers for the purpose of more accurate quantification. HER2-HER3 heterodimers were detected as single fluorescent dots in z-series of cells imaged with confocal microscopy. Additionally, cell nuclei were fluorescently stained with DAPI, and cellular cytoskeletons were labeled with tubulin antibody staining. The image analysis software CellProfiler (Kamentsky et al., 2011 (link)) was used to quantify the PLA signal.
Watson S.S., Dane M., Chin K., Tatarova Z., Liu M., Liby T., Thompson W., Smith R., Nederlof M., Bucher E., Kilburn D., Whitman M., Sudar D., Mills G.B., Heiser L.M., Jonas O., Gray J.W, & Korkola J.E. (2018). Microenvironment-Mediated Mechanisms of Resistance to HER2 Inhibitors Differ between HER2+ Breast Cancer Subtypes. Cell systems, 6(3), 329-342.e6.
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