Human Phospho-SMAD4 (Thr277) and Total SMAD4 PharmaGenie ELISA Kit (SBRS1973)

1. Pre-Coated 96-well Strip Microplate
2. Wash Buffer
3. Anti-Phospho Antibody
4. HRP-Conjugated Secondary Antibody
5. Assay Diluent
6. TMB One-Step Substrate
7. Stop Solution
8. Lysis Buffer
9. Positive Control Sample

Other materials and equipment required:

The Assay Genie Human Phospho-SMAD4 (Thr277) and Total SMAD4 PharmaGenie ELISA Kit (SBRS1973) will require other equipment and materials to carry out the assay. Please see list below for further details.

• Distilled or deionized water
• 100 ml and 1 liter graduated cylinders
• Tubes to prepare sample dilutions
• Protease and Phosphatase inhibitors
• Precision pipettes to deliver 2 ul to 1 ml volumes
• Adjustable 1-25 ml pipettes for reagent preparation
• Benchtop rocker or shaker
• Microplate reader capable of measuring absorbance at 450 nm

1. Prepare all reagents and samples as instructed in the manual.
2. Add 100 ul of sample or positive control to each well.
3. Incubate 2.5 h at RT or O/N at 4 °C.
4. Add 100 ul of prepared primary antibody to each well.
5. Incubate 1 h at RT.
6. Add 100 ul of prepared 1X HRP-Streptavidin to each well.
7. Incubate 1 h at RT.
8. Add 100 ul of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 ul of Stop Solution to each well.
11. Read at 450 nm immediately.