Human Phospho-RB (T826) PharmaGenie ELISA Kit (SBRS1945)

From Assay Genie, this Phospho-RB (T826) ELISA Kit is a colorimetric immunoassay designed to detect human Retinoblastoma protein (RB1, Gene ID 5925; UniProt P06400) phosphorylated at Threonine-826, with listed synonyms including Retinoblastoma-associated protein, p105-Rb, pRb, Rb and pp110. The kit is reactive with human samples and is intended for use with serum, plasma and lysate samples.

Provided components include a Pre-Coated Strip Microplate, Wash Buffer, Anti-Phospho Antibody, HRP-Conjugated Secondary Antibody, Assay Diluent, TMB One-Step Substrate, Stop Solution, Lysis Buffer and a Positive Control Sample. Materials required but not supplied include distilled or deionized water, 100 ml and 1 liter graduated cylinders, tubes to prepare sample dilutions, protease and phosphatase inhibitors, precision pipettes covering 2 µl to 1 ml volumes, adjustable 1–25 ml pipettes for reagent preparation, a benchtop rocker or shaker, and a microplate reader capable of measuring absorbance at 450 nm.

The manufacturer's protocol involves adding 100 µl of sample or positive control to each well and incubating 2.5 h at room temperature or overnight at 4 °C, followed by 100 µl of prepared primary antibody (1 h at room temperature), 100 µl of prepared 1X HRP-Streptavidin (1 h at room temperature), 100 µl of TMB One-Step Substrate Reagent (30 min at room temperature), and 50 µl of Stop Solution, with absorbance read immediately at 450 nm. Validation examples cited by the manufacturer include Jurkat cells treated or untreated with PMA, and HT29 cells treated with Thymidine and Nocodazole, solubilised at 4 × 10⁷ cells/ml in Cell Lysate Buffer. Upon receipt the kit should be stored at −20 °C and used within six months of the date of shipment.