Total-SMARCA4 assay principle

The HTRF Total-SMARCA4 assay quantifies the expression level of SMARCA4 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-SMARCA4 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 SMARCA4 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-SMARCA4 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-SMARCA4 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-SMARCA4 one-plate assay protocol

Detection of Total-SMARCA4 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.

Specificity of HTRF Total SMARCA4 assay using knockout HAP1 cell lines

SMARCA4 expression level was assessed with the HTRF total SMARCA4 kit in HAP1 cells (WT) and different HAP1 cell lines Knocked-Out for SMARCA4 or SMARCA2. Cell density was optimized beforehand to ensure HTRF detection within the dynamic range of the kit (data not shown).

The different cell lines were cultured in a 96-well plate (200,000 cells/well) for 24 hours at 37°C, 5% CO2. After culture medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #1 (1X), then 16 µL of cell lysate were transferred into a low volume white microplate followed by 4 µL of premixed detection reagents. The HTRF signal was recorded after an overnight incubation at RT.
 

In HAP1 KO SMARCA4 cells, the HTRF signal was equivalent to the non-specific signal (dotted line), indicating a complete SMARCA4 gene silencing, whereas the SMARCA4 level was well detected in the other cell line, as expected.

Note that similar expression levels for SMARCA4 in WT and KO SMARCA2 cell lines demonstrate the selectivity of the HTRF SMARCA4 kit over SMARCA2, despite these two proteins sharing more than 70% sequence homology.

Catalog cell line references (Horizon Discovery): HAP1 Wt #C631; HAP1 KO SMARCA4 # HZGHC000922c003; HAP1 KO SMARCA2 # HZGHC004055c008

Validation on various Human and Mouse cell lines

The adherent Human cell lines HeLa (Cervix), HEK293 (Kidney), and the mouse cell line NIH3T3 were plated in 96-well culture plates at a density of 200,000 cells/well and incubated for 24 hours at 37°C, 5% CO2. After culture medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #1 (1X) for 30 min at RT under gentle shaking.

The suspension Human cell lines HEL92.1.7 (erythroleukemia) and MOLT-4 (T lymphoblastic leukemia) were dispensed under 30 µL in a 96-well plate at a density of 200,000 cells/well, incubated for 1h at 37°C, 5% CO2, and lysed with 10 µL of supplemented lysis buffer #1 (4X) for 30 min at RT under gentle shaking.

The SMARCA4 expression levels were assessed with the HTRF Total SMARCA4 kit. Briefly, 16 µL of cell lysate were transferred into a low volume white microplate, followed by 4 µL of premixed HTRF detection reagents. The HTRF signal was recorded after an overnight incubation at RT. The dotted line corresponds to the non-specific HTRF signal. Note that the cell density was optimized beforehand to ensure HTRF detection within the dynamic range of the kit (data not shown).

the HTRF Total SMARCA4 assay efficiently detects endogenous SMARCA4 in various cellular models expressing different levels of the protein.

HTRF total SMARCA4 assay compared to Western Blot

HEL92.1.7 cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After 24h incubation and cell medium removal, the cells were lysed with 3 mL of supplemented lysis buffer #1 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer#1, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF total SMARCA4 detection reagents.

Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

In these conditions, the HTRF total SMARCA4 assay was 4-fold more sensitive than the Western Blot technique.

SMARCA4 Signaling Pathway

SMARCA2 and SMARCA4 are catalytic subunits within the SWI/SNF complex involved in DNA transcription and repair. SMARCA2 and SMARCA4 comprise 6 different domains, including a DNA-dependent ATPase domain and a Bromo domain. The Bromo domain and the H3K27 acetylated levels regulate the binding between DNA and the SWI/SNF complex, while the ATPase domain catalyzes the separation of the two DNA strands. These functions are counteracted by the EZH2 catalytic subunit of the PRC2 complex repressing gene transcription. The loss of function of SMARCA2 or SMARCA4 leads to increased DNA damage, as PRC2 continues to bind to the nucleosome and inhibit DNA repair.