AlphaLISA SureFire Ultra Human and Mouse Total Mitofusin 2 Detection Kit, 10,000 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.

Knockout validation of Mitofusin 2 Total assay

Mitofusin 2 levels were assessed in HEK293 cells (WT) and MFN2 KO (Abcam, ab260861) cells seeded at decreasing cell density in a 96-well plate in complete medium, and incubated overnight at 37°C, 5% CO₂.

The cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Mitofusin 2 levels were then evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximate number of cells is indicated) 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, Mitofusin 2 was only detected in WT cells confirming the specificity of the assay.

Mitofusin 2 expression in various cell lines

Adherent cells were grown to confluency in a T175 flask at 37°C, 5% CO₂ and lysed with Lysis Buffer at a density of 0.5 x 10⁶ cells/mL.

Mitofusin 2 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (5,000 cells) were 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.

Mitofusin 2 expression was detected in a wide range of human and mouse cell lines

PBMCs were isolated from healthy donors and lysed with Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Lysates were serially diluted in Lysis Buffer and Mitofusin 2 levels were then evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximate number of cells is indicated) 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.

Mitofusin 2 expression was detected in PBMCs in less than 500 cells/datapoint.

Cell lysate was prepared from HeLa cells cultured to confluency in a T175 flask lysed in 8 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and Mitofusin 2 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate 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.

Approximate number of cells is indicated. The dotted line represents assay background. The Mitofusin 2 assay has a broad dynamic range and can detect Mitofusin 2 expression in less than 100 cells/datapoint.