Description
MutS Homolog 3 (MSH3) is a DNA mismatch repair protein that forms the MutSβ complex with MSH2 to recognize and repair insertion-deletion loops (IDLs) and larger mismatches in DNA. MSH3 is particularly important for repairing slipped-strand intermediates that arise during DNA replication in repetitive sequences, including microsatellites. The MutSβ complex initiates repair by recruiting MLH1-PMS2 (MutLα) and other downstream factors to excise and resynthesize the error-containing DNA strand. Deficiency in MSH3 leads to microsatellite instability (MSI) and increased mutation rates, particularly in repetitive sequences. MSH3 mutations are associated with certain hereditary cancer syndromes and contribute to tumorigenesis through genomic instability. In cancer therapy, MSH3 status influences response to DNA-damaging agents and immunotherapy, as MSI tumors often exhibit enhanced immunogenicity.
The AlphaLISA SureFire Ultra Human Total MSH3 is a sandwich immunoassay for the quantitative detection of total MSH3 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 MSH3 Total assay
MSH3 levels were assessed in A549 wild type (WT) and A549 KO (Abcam, ab288875) cell lines cultured to confluency in T175 flasks at 37°C, 5% CO2.
Each flask was lysed in 2 mL of Lysis Buffer for 10 minutes at RT with shaking. Lysates were serially diluted in Lysis Buffer and MSH3 levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate was transferred into a 384-well white OptiPlate, followed by 5 µL Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was 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.
MSH3 was detected in WT but not in the KO cells. This confirms the specificity of the assay for the detection of MSH3 protein.
Endogenous MSH3 expression in various cell lines
Adherent 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. Suspension cells were washed with HBSS and lysed with Lysis Buffer at 4 x 106 cells/mL.
MSH3 levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (4,000 adherent cells or 40,000 suspension 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.
Endogenous levels of MSH3 were detected in various cell types.
