Total-RBM39 assay principle

The HTRF Total-RBM39 assay quantifies the expression level of RBM39 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-RBM39 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of RBM39 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

Total-RBM39 two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Total-RBM39 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-RBM39 one-plate assay protocol

Detection of Total-RBM39 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Effect of indisulam using HTRF total RBM39 and α-tubulin detection kits

HeLa cells were plated in a 96-well culture-treated plate (50,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with a dose-response of Indisulam for 6h at 37°C, 5% CO2. The medium was then removed, and the cells were lysed with 50 µl of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking.

After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total-RBM39 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

In parallel, 4 µL of cell lysate (neat or prediluted in the supplemented lysis buffer) were transferred into a low volume white microplate. For HTRF detection, 12 µL of kit diluent were added, followed by the dispensing of 4 µL of premixed HTRF alpha-tubulin detection antibodies. The HTRF signal was also recorded after an overnight incubation at RT.

The anticancer agent indisulam inhibits cell proliferation by causing the degradation of RBM39. The results showed a decrease in the RBM39 signal with increasing concentrations of indisulam, while the alpha-tubulin signal remained stable. This demonstrates that the signal decrease resulted from RBM39 degradation.

Total RBM39 assay specificity

HeLa cells were plated at 25,000 cells per well in a 96-well plate.

After an overnight incubation at 37°C, 5% CO2, the HeLa cells were treated with 25 nM of ON-TARGETplus siRNA (Horizon Discovery) specifically targeting RBM39, or with a non-targeting siRNA (included as a control). After an overnight incubation at 37°C, 5% CO2, the medium was changed for a complete culture medium, and then the cells were incubated for an additional 24h at 37°C.

After the incubation, the cells were lysed with 50 µL of supplemented lysis buffer #4 (1X). The cell lysate was diluted 2 fold and then 16 µL of lysates were transferred into a low volume white microplate before the addition of 4 µL of premixed HTRF Total RBM39 detection antibodies. The HTRF signal was recorded after an overnight incubation at RT.

Cell treatment with RBM39 siRNA led to a significant downregulation of the Total RBM39 signal, with a 62% signal decrease compared to the cells transfected with the non-targeting SiRNA, thus demonstrating the specificity of the kit.

HTRF RBM39 assay is compatible with human and mouse cell lines.

HeLa cells were cultured in a T175 flask in complete medium at 37°C, 5% CO2, to confluency.

After medium removal, the cells were lysed with 3 mL of supplemented lysis buffer #4 (1x) for 30 min at RT under gentle shaking.

NIH/3T3 cells were cultured in a T175 flask in complete medium at 37°C, 5% CO2, to confluency.

After medium removal, the cells were lysed with 3 mL of supplemented lysis buffer #4 (1x) for 30 min at RT under gentle shaking.

Serial dilutions of the cell lysates were performed using supplemented lysis buffer #4 (1x), and 16µL of pure sample and each dilution were transferred into a 384-well small volume microplate before the addition of 4µL of HTRF Total RBM39 detection reagents. Signals were recorded overnight.

The HTRF total RBM39 assay is able to detect mouse RBM39 with a very good signal to background compared to human samples.

HTRF RBM39 assay compared to Western Blot

HeLa cells were cultured in a T175 flask in complete medium at 37°C, 5% CO2, to confluency.

After medium removal, the cells were lysed with 3 mL of supplemented lysis buffer #4 (1x) for 30 min at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer #4 (1x), and 16µL of pure sample and each dilution were transferred into a 384-well small volume microplate before the addition of 4µL of HTRF Total RBM39 detection reagents. Signals were recorded overnight.

Equal amounts of lysates were loaded into a gel for a side by side comparison between HTRF and Western Blot.

In these conditions, the HTRF total RBM39 assay is at least 8-fold more sensitive than the Western Blot.