RayBio® Phospho-Tau (Thr181) Quantitative ELISA kit is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in Human, Mouse & Rat cell lysates. By determining phosphorylated Tau protein in your experimental model system, you can verify pathway activation in your cell lysates. You can simultaneously measure numerous different cell lysates without spending excess time and effort in performing a Western Blotting analysis.

This Sandwich ELISA kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of Human, Mouse & Rat phospho-Tau. An anti-pan Tau antibody has been coated onto a 96-well plate. Standard and Samples are pipetted into the wells and Tau present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-phospho-Tau (Thr181) antibody is used to detect phosphorylated Tau. 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 Tau (Thr181) bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

• Rapidly measure phosphorylated protein in lysates
• Screen numerous different cell lysates without performing a Western Blot analysis
• Minimal hands-on time, convenient, and non-radioactive material

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Anti-Phospho Antibody
• HRP-Conjugated Secondary Antibody
• Assay Diluent
• TMB One-Step Substrate
• Stop Solution
• Lysis Buffer
• Positive Control Sample

• 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 µl 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 µl of sample or positive control to each well.
3. Incubate 2.5 h at RT or O/N at 4 °C.
4. Add 100 µl of prepared primary antibody to each well.
5. Incubate 1 h at RT.
6. Add 100 µl of prepared 1X HRP-Streptavidin to each well.
7. Incubate 1 h at RT.
8. Add 100 µl of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 µl of Stop Solution to each well.
11. Read at 450 nm immediately.

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Upon receipt, the kit should be stored at -20°C. Please use within 6 months from the date of shipment. After initial use, Wash Buffer Concentrate (Item B), Assay Diluent (Item E), TMB One-Step Substrate Reagent (Item H), Stop Solution (Item I) and Cell Lysate Buffer (Item J) should be stored at 4°C to avoid repeated freeze-thaw cycles. Return unused wells to the pouch containing desiccant pack, reseal along entire edge, and store at -20°C. Item D, store at 2-8°C for up to one month (store at -20°C for up to 6 months, avoid repeated freeze-thaw cycles). Reconstituted Positive Control (Item K) should be stored at -70°C.