The RayBio^® Ubiquitinated p53 Quantitative ELISA kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of Human, Mouse, Rat Ubiquitinated p53. An anti-pan p53 antibody has been coated onto a 96-well plate. Standard and Samples are pipetted into the wells and p53 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-ubiquitin antibody is used to detect ubiquitinated p53. After washing away unbound antibody, HRP-conjugated streptavidin is pipetted into the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of ubiquitinated p53 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

• Quantify quickly and easily
• Screen numerous different lysates without performing a Western Blot analysis
• Minimal hands-on time, convenient, and non-radioactive material

• Microplate reader capable of measuring absorbance at 450 nm
• Protease and Phosphatase inhibitors
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Adjustable 1-25 mL pipettes for reagent preparation
• 100 mL and 1 liter graduated cylinders
• Log-log graph paper or computer and software for ELISA data analysis
• Absorbent paper
• Tubes to prepare positive control or sample dilutions
• Distilled or deionized water
• Shaker

1. Prepare all reagents, samples and standards as instructed.
2. Add 100 µL of each standard and sample to each well. Incubate 2.5 hours at room temperature or overnight at 4ºC with gentle shaking.
3. Add 100 µL prepared detection antibody to each well. Incubate for 1 hour at room temperature with gentle shaking.
4. Add 100 μL prepared HRP-Streptavidin solution. Incubate for 1 hour at room temperature with gentle shaking.
5. Add 100 μL TMB One-Step Substrate Reagent to each well. Incubate 30 minutes at room temperature.
6. Add 50 μL Stop Solution to each well. Read at 450 nm immediately.

The entire kit may be stored at -20°C for up to 6 months from the date of shipment. Avoid repeated freeze-thaw cycles.