Protein Name & Synonyms:
Mitogen-activated protein kinase kinase kinase 7 (EC 188.8.131.52) (Transforming growth factor-beta-activated kinase 1) (TGF-beta-activated kinase 1)
- MAPK Signaling
- NFkB Signaling
This ELISA kit recognizes Human TAK1 phosphorylated at site Serine-412.
Number of Targets Detected:
Compatible Sample Types:
- Cell Lysates
- Tissue Lysates
Method of Detection:
1, 2, or 5 x 96-Well Strip Microplate Kit
- 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
- 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
- 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.
HeLa cells were treated with Calyculin A. 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.
Calyculin A/IL-1 beta Stimulation of HeLa Cell Line
HeLa cells were treated with Calyculin A and IL-1 beta. Cell lysates were treated or untreated with Lambda Protein Phosphatase (LPP) and analyzed using this phosphoELISA and Western Blot.
Upon receipt, the kit should be stored at –20°C. Please use within 6 months from the date of shipment.