{"product_id":"revvity-alsu-psm3-b500","title":"Revvity, ALSU-PSM3-B500, AlphaLISA Human and Mouse High Specificity Phospho-SMAD3 (Ser423\/425) Detection Kit, 500 Assay Points","description":"\u003cp\u003eOverview\u003cbr\u003e\nSMAD3 is an intracellular signal transducer and transcription factor that mediates TGF-β and activin signaling to regulate cell growth, differentiation, and extracellular matrix production. Upon TGF-β receptor activation, SMAD3 is phosphorylated, enabling formation of complexes with SMAD4 that translocate to the nucleus. The SMAD3\/SMAD4 complex regulates transcription of genes including collagens, fibronectin, and cyclin-dependent kinase inhibitors. SMAD3 plays context-dependent roles as both a tumor suppressor and tumor promoter. Loss of SMAD3 contributes to colorectal cancer, while excessive SMAD3 activation drives pathological fibrosis in multiple organs.\u003cbr\u003e\nThe AlphaLISA SureFire Ultra Human and Mouse High Specificity Phospho-SMAD3 (Ser423\/425) is a sandwich immunoassay for the quantitative detection of phospho-SMAD3 (Ser423\/425)  in cellular lysates, using Alpha Technology.\u003cbr\u003e\nFormats:\u003c\/p\u003e\n\n\u003cp\u003eThe HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.\u003cbr\u003e\nThe 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.\u003cbr\u003e\nThe 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.\u003cbr\u003e\nThe 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.\u003c\/p\u003e\n\n\u003cp\u003eAlphaLISA SureFire Ultra kits are compatible with:\u003c\/p\u003e\n\n\u003cp\u003eCell and tissue lysates\u003cbr\u003e\nAntibody modulators\u003cbr\u003e\nBiotherapeutic antibodies\u003c\/p\u003e\n\n\u003cp\u003eAlphaLISA SureFire Ultra kits can be used for:\u003c\/p\u003e\n\n\u003cp\u003eCellular kinase assays\u003cbr\u003e\nReceptor activation studies\u003cbr\u003e\nHigh-throughput screening for preclinical studies\u003cbr\u003e\nHow it works\u003cbr\u003e\nPhospho-AlphaLISA SureFire Ultra assay principle\u003cbr\u003e\nThe Phospho-AlphaLISA SureFire Ultra assay measures a protein target when phosphorylated at a specific residue.\u003cbr\u003e\nThe assay uses two antibodies which recognize the phospho epitope and a distal epitope 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 phosphorylated 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 phosphoprotein present in the sample.\u003c\/p\u003e\n\n\n  \u003cbr\u003e\n        \n\n\n\t\u003cbr\u003e\n  \u003cbr\u003e\n        \u003cbr\u003e\n  \n\n      \u003cbr\u003e\n  \u003cbr\u003e\n  \u003cbr\u003e\n    \u003cbr\u003e\n      \u003cbr\u003e\n        \u003cbr\u003e\n        \u003cbr\u003e\n        \u003cbr\u003e\n        \u003cbr\u003e\n      \u003cbr\u003e\n    \n\n    \u003cbr\u003e\n      \n\n      \u003cbr\u003e\n        \n\n      \u003cbr\u003e\n    \n\n  \n\n\n\n\n\u003cp\u003e \u003c\/p\u003e\n\n\n\u003cp\u003ePhospho-AlphaLISA SureFire Ultra two-plate assay protocol\u003cbr\u003e\nThe 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 Phospho-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.\u003c\/p\u003e\n\n\n\n\n  \u003cbr\u003e\n        \n\n\n\t\u003cbr\u003e\n  \u003cbr\u003e\n        \u003cbr\u003e\n  \n\n      \u003cbr\u003e\n  \u003cbr\u003e\n  \u003cbr\u003e\n    \u003cbr\u003e\n      \u003cbr\u003e\n        \u003cbr\u003e\n        \u003cbr\u003e\n        \u003cbr\u003e\n        \u003cbr\u003e\n      \u003cbr\u003e\n    \n\n    \u003cbr\u003e\n      \n\n      \u003cbr\u003e\n        \n\n      \u003cbr\u003e\n    \n\n  \n\n\n\n\n\n\n\n\n\u003cp\u003ePhospho-AlphaLISA SureFire Ultra one-plate assay protocol\u003cbr\u003e\nDetection of Phosphorylated 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.\u003cbr\u003e\nAssay sensitivity\u003cbr\u003e\nAssay sensitivity - cell lysate\u003cbr\u003e\nCell lysate was prepared from RAW 264.7 cells cultured to confluence in a T175 flask and stimulated with 50 ng\/mL TGF-β for 90 minutes. Cells were lysed with 6 mL of Lysis Buffer for 10 minutes at RT with shaking.\u003cbr\u003e\nLysate was serially diluted in Lysis Buffer and SMAD3 Phospho (Ser423\/425) and Total levels were evaluated using AlphaLISA SureFire Ultra High Specificity assays. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. 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.\u003cbr\u003e\nApproximate number of cells per datapoint is indicated. The dotted line represents assay background. The assay can detect SMAD3 Phospho (Ser423\/425) down to 2,500 cells\/datapoint. \u003cbr\u003e\nApplication-Cell Signaling\u003cbr\u003e\nAutomation Compatible-Yes\u003cbr\u003e\nBrand-AlphaLISA SureFire Ultra\u003cbr\u003e\nDetection Modality-Alpha\u003cbr\u003e\nProtocol Time-2h at RT\u003cbr\u003e\nSample Volume-10 µL\u003cbr\u003e\nShipping Conditions-Shipped in Blue Ice\u003cbr\u003e\nTarget-SMAD3\u003cbr\u003e\nTarget Class-Phosphoproteins\u003cbr\u003e\nTarget Species-Human Mouse\u003cbr\u003e\nTechnology-Alpha\u003cbr\u003e\nTherapeutic Area-Inflammation NASH\/Fibrosis Oncology\u003cbr\u003e\nUnit Size-500 Assay Points\u003c\/p\u003e","brand":"Revvity","offers":[{"title":"Default Title","offer_id":46774656991401,"sku":"ALSU-PSM3-B500","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/revvity-alsu-psm3-b500","provider":"Iright","version":"1.0","type":"link"}