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| Solution Information | help | |
| Enzyme: | Mothers against decapentaplegic homolog 3 | |
| inhibitor: | BDBM41957 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Compounds identified as active from the primary screen, AID 630 - HTS of SMAD transcription factor inhibitors, were selected for testing in this assay. Reagent: 1. Wash Buffer: Tris-buffer Saline (25 mM Tris, 150mM NaCl; pH 7.2), 0.1% BSA, 0.05% Tween-20. 2. Pierce# 15509 (white 96-well) or #15512 (white 384-well) Racti-Bind NeutrAvidin High Binding Capacity Coated Plates. 3. Rockland-# 600-406-382 Biotin Conjugated Affinity purified anti-6X His epitope tag 100ug. 4. Roche# 120138190001. Anti HA Peroxidase, high affinity 3F10. 5. Detection Reagent- Pierce #37070, SuperSignal @ ELISA Pico Chemiluminescent Substrate. 6. Smad3P-Trimer/Monomer purified 1.3ug/ul. Molecular Weight 48.1 kilodaltons (Stored at -80 deg C). 7. HA-Lysate- HEK-293 cells (ATCC) grown in 100x20mm culture dishes and transfected at 50% confluence. Harvested after 24 hours(at approx. 90% confluent) in 1 ml of lysis buffer; Lysate is flash frozen in liquid nitrogen and stored at -80oC Procedures: 1. Wash each wel | |
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