BacMam-enabled Cellular Assays are an efficient, easy-to-optimize, and robust method for interrogating specific signal transduction events in a cell background of choice. BacMam-enabled Cellular Assays combine two robust technologies: BacMam-mediated gene delivery and LanthaScreen® TR-FRET technology. Each technology provides unique benefits, including portability into different cell types and an assay readout that is quick and amenable to high-throughput applications.
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.
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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Interested in custom BacMam virus generation or building an assay for your target of interest? Custom project scopes can range from something as minimal as creating a BacMam virus from your gene of interest to full assay development and batch transduction of your BacMam virus into your cell line of choice.
BacMam-enabled Cellular Assay evaluation kits provide enough reagents for ~800 assays (in a 384-well plate format). Kits include the following reagents:
- BacMam reagent
- Terbium-labeled antibody
- Lysis buffer
All kit reagents are available in bulk sizing.
For Research Use Only. Not for use in diagnostic procedures.