Human Phospho-MLKL (S345) and Total MLKL ELISA

RayBio® Human Phospho-MLKL (Ser345) and Total MLKL ELISA Kit. This assay semi-quantitatively measures MLKL phosphorylated at Serine-345 as well as total MLKL in lysate samples.

$541.00  
PEL-MLKL-S345-T-1
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Antigen Information

Accession Number:
  • Q8NB16
Gene ID:
  • 197259
Gene Symbols:
  • MLKL
Protein Name & Synonyms:
Mixed lineage kinase domain-like protein (hMLKL)

Assay Format

Pathway:
  • Necroptosis
Specificity:
The antibody pair provided in this kit recognizes Human MLKL phosphorylated at site Serine-345 as well as total MLKL
Number of Targets Detected:
2
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

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
  • Anti-Phospho Antibody
  • HRP-Conjugated Secondary Antibody
  • 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
HT29 cells were treated with Calyculin A. Solubilize cells at 4 x 10^7 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.
Calyculin A Stimulation of HT29 Cell Line
HT29 cells were treated with Calyculin A. Cell lysates were analyzed using this phosphoELISA and Western Blot.


Storage/Stability

Upon receipt, the kit should be stored at –20°C. Please use within 6 months from the date of shipment.
This Human Phospho-MLKL (Ser345) ELISA kit is used to detect MLKL activation in the designated cancer cell lines under certain treatment conditions. We tried this kit with less number of cells than recommended number of cells and it works fine. Seems it is a very sensitive assay kit.

NIH
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Usage

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

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