Description
Mitofusins (MFN1 and MFN2) are dynamin-related GTPases in the outer mitochondrial membrane that mediate mitochondrial fusion, essential for maintaining mitochondrial morphology and function. Mitofusins function as molecular tethers that bring adjacent mitochondria into close proximity, with GTP hydrolysis driving membrane fusion. MFN1 exhibits higher fusion activity while MFN2 has additional roles in ER-mitochondria tethering. Mutations in MFN2 cause Charcot-Marie-Tooth disease type 2A, a peripheral neuropathy from impaired mitochondrial dynamics. Altered mitofusin function is implicated in metabolic diseases, neurodegenerative disorders, and cancer.
The AlphaLISA SureFire Ultra Human and Mouse Total Mitofusin 2 is a sandwich immunoassay for the quantitative detection of total Mitofusin 2 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
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the target 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 target 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 for 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 enables cell 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 robust 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
Mitofusin 2 assay sensitivity
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.
