HTRF Human & Mouse Phospho-SMAD4 (Thr277) Detection Kit, 500 Assay Points

Phospho SMAD4 (Thr277) cell line compatibility

Hela and HAPI cell lines were selected to test human compatibility, while NIH 3T3 cells were chosen for mouse compatibility. 25,000, 50,000, and 100,000 cells of these different cell lines were plated in 96-well  culture plates. After a 48h incubation at 37°C, 5% CO2, the cell culture medium was removed and 50µL of lysis buffer were added to the wells. A lysis step was carried out, shaking gently for 30 minutes. 16µL of the pure samples diluted ½ were transferred into a 384-well small volume plate, then 4µL of Phospho T277 SMAD4 HTRF detection reagents were added. Signals were recorded overnight.

This data enables better cell density selection for the different cell lines. The HTRF Phospho SMAD4 (Thr277) assay is able to detect human as well as mouse phosphorylated SMAD4.

Phospho SMAD4 (Thr277) assay specificity

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

After an overnight incubation at 37°C, 5% CO2, the HAPI wt cells were treated with 25 nM of ON-TARGETplus siRNA (Horizon Discovery) targeting specifically SMAD2, SMAD3, and SMAD4, 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.

HAPI SMAD4 KO cells (Horizon Discovery)were plated at 25,000 cells per well in a 96-well plate and were incubated for 4 days.

After the incubation, the cells were lysed with 50 µL of supplemented lysis buffer #4 (1X), and 16 µL of lysates were transferred into a low volume white microplate before the addition of 4 µL of premixed HTRF Phospho T277 SMAD4 detection antibodies. The HTRF signal was recorded after an overnight incubation at RT.

Cell treatment with SMAD4 siRNA led to a significant downregulation of total SMAD4, leading to a 61% Phosphorylation decrease compared to the cells transfected with the non-targeting siRNA. Moreover, no HTRF signal was observed in the HAPI SMAD4 KO cell lysate .

On the other hand, no decrease in signal was observed with cells treated with SMAD2 or SMAD3 SiRNA , demonstrating the specificity of the kit. The downregulation of SMAD2 & SMAD3 was checked using our HTRF Total SMAD2 & SMAD3 detection kits, showing a 60% and 66% loss of signal respectively.

HTRF Phospho SMAD4 (Thr277) Assay compared to Western Blot

 

 

 

HTRF Phospho SMAD4 (Thr277) Assay compared to Western Blot

HAPI cells were cultured in complete medium at 37°C, 5% CO2 in a T175 flask to 80% confluency.

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 Phospho T277-SMAD4 HTRF 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 phospho SMAD4 (Thr277) assay is at least 4-fold more sensitive than the Western Blot.