Human Phosphotyrosine EphB4 PharmaGenie ELISA Kit (SBRS1812)

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SKU:
SBRS1812
Product Type:
ELISA Kit
Reactivity:
Human
Applications:
ELISA
ELISA Type:
Sandwich

Description

Human Phosphotyrosine EphB4 PharmaGenie ELISA Kit (SBRS1812)

Assay Genie Phospho-EphB4 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-EphB4 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-EphB4. An anti-EphB4 antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and phosphorylated and unphosphorylated EphB4 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-EphB4 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

Product SKU:SBRS1812
Size:96T
Application:The antibody pair provided in this kit recognizes Human Tyrosine-Phosphorylated-EphB4.
Uniprot:P54760
Gene ID:2050
Gene Names:EPHB4 / HTK / MYK1 / TYRO11
Pathway:Tyrosine Kinase Family
Synonyms:Ephrin type-B receptor 4 (EC 2.7.10.1) (Hepatoma transmembrane kinase) (Tyrosine-protein kinase TYRO11)
Target Species:Human
Compatible Sample Types:Cell Lysates, Tissue Lysates
Design Principle:Sandwich-based
Method of Detection:Colorimetric
Quantitative/Semi-Quantitative:Semi-Quantitative
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.
  1. Pre-Coated 96-well Strip Microplate
  2. Wash Buffer
  3. Biotinylated Anti-Phosphotyrosine Antibody
  4. Stop Solution
  5. Assay Diluent(s)
  6. Positive Control Sample
  7. Lysis Buffer
  8. Streptavidin-Conjugated HRP
  9. TMB One-Step Substrate

Other materials and equipment required:

The Assay Genie Human Phosphotyrosine EphB4 PharmaGenie ELISA Kit (SBRS1812) will require other equipment and materials to carry out the assay. Please see list below for further details.

  • 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 ul 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 ul of sample or positive control to each well.
  3. Incubate 2.5 h at RT or O/N at 4 °C.
  4. Add 100 ul of prepared primary antibody to each well.
  5. Incubate 1 h at RT.
  6. Add 100 ul of prepared 1X HRP-Streptavidin to each well.
  7. Incubate 1 h at RT.
  8. Add 100 ul of TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 ul of Stop Solution to each well.
  11. Read at 450 nm immediately.
Product ImageZR751 cells were untreated or treated with b-estradiol for 1 hour. Cell lysates were analyzed using this phosphoELISA and Western Blot.
Product ImageJurkat 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.
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