Human/Mouse/Rat Phospho-MKK3 (S189) ELISA

RayBio® Human/Mouse/Rat Phospho-MKK3 (S189) ELISA Kit. This assay semi-quantitatively measures phosphorylated MKK3 (Ser189) in lysate samples.

$1,984.00  
PEL-MKK3-S189-5
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Antigen Information

Accession Number:
  • P46734
Gene ID:
  • 5606
Gene Symbols:
  • MAP2K3
  • MEK3
  • MKK3
  • PRKMK3
  • SKK2
Protein Name & Synonyms:
Dual specificity mitogen-activated protein kinase kinase 3 (MAP kinase kinase 3) (MAPKK 3) (EC 2.7.12.2) (MAPK/ERK kinase 3) (MEK 3) (Stress-activated protein kinase kinase 2) (SAPK kinase 2) (SAPKK-2) (SAPKK2)

Assay Format

Pathway:
  • MAPK Signaling
Specificity:
The antibody pair provided in this kit recognizes human, mouse, and rat PEL phosphorylated at site Serine-189.
Number of Targets Detected:
1
Species Detected:
  • Human
  • Mouse
  • Rat
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-MKK3 (S189) 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 and rat cell lysates. By determining phosphorylated MKK3 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 measurement of human, mouse and rat phospho-MKK3. An anti-pan MKK3 antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and MKK3 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and rabbit anti-MKK3 (S189) antibody is used to detect phosphorylated MKK3. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG 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 MKK3 (S189) bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

Product Features

  • Simultaneously measure Phosphorylated protein and pan protein in one experiment (for normalization purpose)
  • 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
  • Anti-Phospho Antibody
  • Anti-Pan Antibody
  • HRP-Conjugated Secondary Antibody
  • Streptavidin-Conjugated HRP
  • Assay Diluent
  • TMB One-Step Substrate
  • Stop Solution
  • Lysis Buffer
  • Positive Control Sample
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
HeLa cells were treated with UV. Solubilize cells at 4 x 107 cells/ml in Cell Lysate Buffer. Serial dilutions of lysates were analyzed in this ELISA. Please see step 3 of Part VI Reagent Preparation for detail.
MKK3 ELISA Pos Control
UV Stimulation of HeLa Cell Line
HeLa cells were treated or untreated with UV. Cell lysates were analyzed using this phosphoELISA and Western Blot.

MKK3 (S189) - Western Blot

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), 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.

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Usage

This product is furnished for LABORATORY RESEARCH USE ONLY.
Not for diagnostic or therapeutic use.

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