BacMam-enabled Cellular Assays

Compared to other types of cellular assays, BacMam-enabled Cellular Assays reduce assay development time by minimizing the need to generate a stable cell line while also allowing choice of cellular background. Once cells are transduced with the BacMam reagent, LanthaScreen® technology is utilized for the readout. This assay format has a simple, “addition only” protocol requiring minimal hands-on time ( Figure 1)

Figure 1.  BacMam-enabled Cellular Assay work flow. Cells are treated with BacMam reagent encoding a GFP-fusion protein and plated in 384-well format. 24 hours post-transduction, the cells are stimulated to induce the posttranslational modification of the GFP-substrate (e.g. phosphorylation as shown). Cells are then lysed in the presence of a terbium anti-modification-specific antibody prior to the LanthaScreen® assay readout.

Cell types successfully transduced with BacMam reagents include numerous transformed cell lines (e.g. U2-OS, HEK 293, and HeLa), many primary cells (e.g., fibroblasts, endothelial, and epithelial cells), and stem cells (e.g. adipocyte derived and mesenchymal stem cells). These cells have been shown to be efficiently transduced without adverse affects (Figure 2).

For more information on BacMam compatible cell types, visit www.invitrogen.com/bacmam

Figure 2. BacMam-enabled Cellular Assays for detecting Histone H3 acetylation at Lysine 9 in various cell backgrounds. (A) 786-0; (B) U2-OS; (C) HCT-116. Cells were transduced with BacMam Histone H3 reagent and plated onto a 384-well assay plate in low-serum assay medium overnight. Cells were then treated with indicated amounts of trichostatin A (HDAC inhibitor) for 3 hours prior to performing the LanthaScreen® assay. All three assays had response ratios greater than 3.0. Similar results have been achieved for various cell types using BacMam-enabled Cellular Assays.

Nucleosome functions are highly regulated by a multitude of dynamic posttranslational modifications such as phosphorylation, acetylation, methylation, ubiquitination, and others. The use of LanthaScreen® technology in BacMam-enabled Cellular Assays allows for examination of multiple posttranslation modifications on a given substrate as long as a modification-specific antibody is available. The acetylation of Histone H3 at Lys9 and the cell cycle-dependent phosphorylation of Histone H3 at Ser10 can be detected with terbium-labeled anti-Histone H3 acetyl-Lys9 and anti-phospho Ser10 specific antibodies, respectively (Figures 3 and 4). Bacmam-enabled Cellular Assays enable interrogation of multiple enzymatic activities responsible for posttranslational modifications, including challenging targets like histones.



Figure 3.   BacMam-enabled Cellular Assay for detecting Histone H3 acetylation at Lysine 9. U2-OS cells were transduced with BacMam Histone H3 reagent and  plated onto a 384-well assay plate in low-serum assay medium overnight. Cells were then treated with indicated amounts of HDAC inhibitors for 3 hours prior to performing the LanthaScreen® assay. The BacMam Histone H3 [AcLys9] Cellular Assay can detect known HDAC inhibitors with a broad potency.

Figure 4.   BacMam-enabled Cellular Assay for detecting Histone H3 phosphorylation at Serine 10. U2-OS cells were transduced with BacMam Histone H3 reagent and plated onto a 384-well assay plate in low-serum assay medium overnight. (A) Cells were then treated with indicated amounts of calyculin A for 1 hour prior to performing the TR-FRET assay. (B) Cells were pretreated with aurora kinase inhibitor VX680 for 1 hour and then incubated with calyculin A for an additional hour prior to performing the LanthaScreen® assay. The BacMam Histone H3 [pSer10] Cellular Assay can successfully measure aurora kinase activity.

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