Description
Utrophin is an autosomally-encoded 376-kDa cytoskeletal protein that is similar in structure and function to dystrophin. It is a ubiquitously expressed protein that plays a role in anchoring the cytoskeleton to the plasma membrane. Utrophin is highly expressed in developing muscle, and is enriched at the neuromuscular junction of mature muscle. Duchenne's muscular dystrophy patients lack dystrophin, and utrophin is consequently up-regulated and redistributed to locations normally occupied by dystrophin. HTRF Utrophin kit is designed for its rapid detection in cell lysates.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
Utrophin assay principle
Utrophin is measured using a sandwich immunoassay involving two specific anti-utrophin antibodies, respectively labelled with Europium Cryptate (donor) and d2 (acceptor). The intensity of the signal is proportional to the concentration of utrophin present in the sample.
Utrophin assay protocol
The Utrophin assay features a two-plate assay protocol, where cells are first plated, stimulated, and lysed in the same culture plate. Lysates are then transferred to the assay plate for the detection of utrophin. This protocol enables the cells' viability and confluence to be monitored. The antibodies labelled with HTRF fluorophores may be pre-mixed and added in a single dispensing step to further streamline the assay procedure. The assay detection can be run in 96- to 384-well plates by simply resizing each addition volume proportionally.
Endogenous level of utrophin in HeLa cells
Human HeLa cells were plated under 100 µl in 96-well plates at different cell densities (100K, 50K, 25K, 12.5K, 6.25K, and 3.1K cells/well) in complete culture medium, and incubated at 37°C, 5% CO2. The day after, medium was removed and cells were then lysed with 50 µL of lysis buffer #3 for 30 minutes at room temperature under gentle shaking. Next, 16 µL of lysate were transferred into a low volume white microplate before the addition of 4 µL of the premixed HTRF Utrophin detection reagents. The HTRF signal was recorded after ovenight incubation.
Endogenous level of utrophin in C2C12 cells
Mouse C2C12 cells were plated under 100 µl in a 96-well plates at different cell densities (200K, 100K, 50K, 25K, 12.5K, and 6.25K cells/well) in complete culture medium and incubated at 37°C, 5% CO2. The day after, medium was removed and cells were then lysed with 50 µL of lysis buffer #3 for 30 minutes at room temperature under gentle shaking Next, 16 µL of lysate were transferred into a low volume white microplate before the addition of 4 µL of the premixed HTRF Utrophin detection reagents. The HTRF signal was recorded after overnight incubation.
