Human Phosphotyrosine EphA4 ELISA

RayBio® Human Phosphotyrosine EphA4 ELISA Kit. This assay semi-quantitatively measures phosphotyrosine EphA4 in lysate samples.

Specifications

Introduction

RayBio^® Phospho-EphA4 ELISA kit is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in cell lysates. By determining phospho-EphA4 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 Blot analysis.

This sandwich-based ELISA kit is an in vitro enzyme-linked immunosorbent assay for the measurement of human phospho-EphA4. An anti-EphA4 antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and phosphorylated and unphosphorylated EphA4 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-phosphotyrosine antibody is used to detect only tyrosine-phosphorylated protein. After washing away unbound antibody, HRP-conjugated streptavidin is pipetted to 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 phosphor-EphA4 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

Product Features

• - 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

Application Notes

Kit Components

• - Pre-Coated 96-well Strip Microplate

• - Wash Buffer

• - Biotinylated Anti-Phosphotyrosine Antibody

• - Stop Solution

• - Assay Diluent(s)

• - Positive Control Sample

• - Lysis Buffer

• - Streptavidin-Conjugated HRP

• - TMB One-Step Substrate

Other Materials Required

• - 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

Protocol Outline

• 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.

Typical Data

Positive Control

Jurkat cells were treated with Pervanadate at 37°C for 10 min. Solubilize cells at 4 x 10⁷ cells/ml in lysis buffer. Serial dilutions of lysates were analyzed in this ELISA. Please see step 3 of Part VI Reagent Preparation for details.

Pervanadate Stimulation of Jurkat Cell Line

Jurkat cells were treated or untreated with Pervanadate for 10 min at 37°C. Cell lysates were analyzed using this phosphoELISA.

Storage/Stability

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), HRP-Streptavidin (Item G), 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. Reconstituted Positive Control (Item K) should be stored at -70 °C.

Catalog #: PEL-EPHA4-Y-1 -- 1 Plate Kit

Catalog #: PEL-EPHA4-Y-2 -- 2 Plate Kit

Catalog #: PEL-EPHA4-Y-5 -- 5 Plate Kit

Please email us at cs@medikonia.com for any enquiry. To place an order, please include the catalog number(s) of the product(s) in the email.

Manual

Safety Data Sheet