Overview Myogenin (MYOG) is a muscle-specific transcription factor belonging to the MyoD family of basic helix-loop-helix proteins that regulates skeletal muscle differentiation and regeneration. Myogenin is expressed during the terminal differentiation phase of myogenesis, where it forms heterodimers with E-proteins to bind E-box sequences in muscle-specific gene promoters. It activates expression of structural muscle proteins including myosin heavy chain, troponin, and creatine kinase, driving the transition from proliferating myoblasts to multinucleated myotubes. Myogenin is essential for proper muscle fiber formation and is reactivated during muscle regeneration following injury. Dysregulation of myogenin is implicated in muscle wasting diseases, muscular dystrophies, and rhabdomyosarcoma, where it serves as both a diagnostic marker and potential therapeutic target for restoring muscle function and combating muscle-related pathologies. The AlphaLISA SureFire Ultra Human and Mouse Total Myogenin is a sandwich immunoassay for the quantitative detection of total Myogenin in cellular lysates, using Alpha Technology. Formats: The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume. The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume. The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume. The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume. AlphaLISA SureFire Ultra kits are compatible with: Cell and tissue lysates Antibody modulators Biotherapeutic antibodies AlphaLISA SureFire Ultra kits can be used for: Cellular kinase assays Receptor activation studies High-throughput screening for preclinical studies How it works Total-AlphaLISA SureFire Ultra assay principle The Total-AlphaLISA SureFire Ultra assay measures the expression level of a protein target in a cell lysate. The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the targeted protein. AlphaLISA assays require two bead types: Acceptor and Donor beads. Acceptor beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of targeted protein, the two antibodies bring the Donor and Acceptor beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor bead, allowing the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample. Total-AlphaLISA SureFire Ultra two-plate assay protocol The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Ultra detection reagents. This protocol permits the cells viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets. Total-AlphaLISA SureFire Ultra one-plate assay protocol Detection of Total target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining AlphaLISA SureFire Ultra quality. Assay versatility Basal Myogenin expression in response to culturing conditions RD and C2C12 cells were seeded in a 96-well plate at various cell density (10,000 to 80,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2 . The cells were then lysed with 25 µL to 200 µL/well of Lysis Buffer to achieve the same cells/datapoint. Myogenin and Total ERK levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings. High cell density acts as a signal for the onset of muscle differentiation. As expected, Myogenin expression is increased when cells are seeded at high density. Total ERK levels were unchanged (data not shown). C2C12 cells were seeded in a 96-well plate (5,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2 . The cells were either left in complete DMEM (high serum) or DMEM + 2% horse serum (low serum) for 24 hours. After treatment, the cells were lysed with 25 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Myogenin and Total ERK levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings. Low serum conditions induces the cells to undergo differentiation from myoblasts into myotubes. Myogenin expression is activated in myoblasts as they begin the differentiation process. Total ERK levels were unchanged (data not shown). Myogenin expression in various cell lines Cells were seeded at 40,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO2 . Cells were lysed with 100 µL of Lysis Buffer. Myogenin levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (4,000 adherent cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings. As expected, Myogenin was only expressed in RD and C2C12 cells, both of which are derived from muscle tissue.
