RayBio® Human Phosphotyrosine JAK1 ELISA Kit. This assay semi-quantitatively measures phosphotyrosine JAK1 in lysate samples.

Antigen Information

Accession Number:

P23458

Gene ID:

3716

Gene Symbols:

JAK1
JAK1A
JAK1B

Protein Name & Synonyms:

Tyrosine-protein kinase JAK1 (EC 2.7.10.2) (Janus kinase 1) (JAK-1)

Assay Format

Pathway:

JAK/STAT Signaling
Tyrosine Kinase Family

Specificity:

The antibody pair provided in this kit recognizes Human Tyrosine-Phosphorylated-JAK1.

Number of Targets Detected:

Species Detected:

Human

Compatible Sample Types:

Cell Lysates
Tissue Lysates

Design Principle:

Sandwich-based

Method of Detection:

Colorimetric

Quantitative/Semi-Quantitative:

Semi-Quantitative

Solid Support:

96-well Microplate

Product Specifications

Size:

1, 2, or 5 x 96-Well Strip Microplate Kit

Introduction

RayBio® Phospho-JAK1 ELISA (Enzyme-Linked Immuno-sorbent Assay) 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 phosphor-JAK1 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 ELISA kit is an in vitro enzyme-linked immunosorbent assay for the measurement of human phospho-JAK1. An anti-JAK1 antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and phosphorylated and unphosphorylated JAK1 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 phospho-JAK1 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

Prepare all reagents and samples as instructed in the manual.
Add 100 µl of sample or positive control to each well.
Incubate 2.5 h at RT or O/N at 4 °C.
Add 100 µl of prepared primary antibody to each well.
Incubate 1 h at RT.
Add 100 µl of prepared 1X HRP-Streptavidin to each well.
Incubate 1 h at RT.
Add 100 µl of TMB One-Step Substrate Reagent to each well.
Incubate 30 min at RT.
Add 50 µl of Stop Solution to each well.
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 detail.
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.