AlphaLISA SureFire Ultra Human and Mouse Total Myogenin Detection Kit, 100 Assay Points

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.

Decrease in Myogenin expression in response to BMP-2 treatment

C2C12 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with 50 ng/mL BMP-2 in serum-free medium for the indicated times.

After treatment, the cells were lysed with 100 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 2,000 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.

As expected, BMP-2 treatment decreased the levels of Myogenin in C2C12 cells while Total ERK levels were unchanged (data not shown).

C2C12 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of cycloheximide for 2 hours.

After treatment, the cells were lysed with 100 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.

The inhibition of protein synthesis by cycloheximide resulted in a decrease in the levels of Myogenin.

C2C12 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of H₂O₂ for 2 hours.

After treatment, the cells were lysed with 50 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 8,000 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.

As expected, H₂O₂ treatment causes a suppression of Myogenin expression, Total ERK levels are unchanged.

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.