This antibody gave a positive signal in human thymus and human kidney tissue lysates and in the following cell lines: Jurkat; Ramos; Raji.
This antibody gave a positive result in IHC in the following FFPE tissue: Human normal kidney.
This antibody gave a positive result when used in the following formaldehyde fixed cell lines: DU145
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 116 kDa (predicted molecular weight: 116 kDa).
Use a concentration of 10 µg/ml.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Plays a role in the inducible expression of cytokine genes in T-cells, especially in the induction of the IL-2.
Isoform 1 is predominantly expressed in thymus and is also found in peripheral blood leukocytes and kidney. Isoform 2 is predominantly expressed in skeletal muscle and is also found in thymus, kidney, testis, spleen, prostate, ovary, small intestine, heart, placenta and pancreas. Isoform 3 is expressed in thymus and kidney. Isoform 4 is expressed in thymus and skeletal muscle.
Contains 1 RHD (Rel-like) domain.
Rel Similarity Domain (RSD) allows DNA-binding and cooperative interactions with AP1 factors.
Phosphorylated by NFATC-kinase; dephosphorylated by calcineurin.
Cytoplasm. Nucleus. Cytoplasmic for the phosphorylated form and nuclear after activation that is controlled by calcineurin-mediated dephosphorylation. Rapid nuclear exit of NFATC is thought to be one mechanism by which cells distinguish between sustained and transient calcium signals. The subcellular localization of NFATC plays a key role in the regulation of gene transcription.
ICC/IF image of ab93628 stained DU145 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab93628 at 10µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
IHC image of NFAT4 staining in Human normal kidney formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab93628, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Western blot - NFAT4 antibody (ab93628)
All lanes : Anti-NFAT4 antibody (ab93628) at 1 µg/ml
Lane 1 : Human thymus tissue lysate - total protein (ab30146) Lane 2 : Human kidney tissue lysate - total protein (ab30203) Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate Lane 4 : Ramos (Human Burkitt's lymphoma cell line) Whole Cell Lysate Lane 5 : Raji (Human Burkitt's lymphoma cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
NFAT4 has six isoforms (Swissprot data). Ab93628 is predicted to cross react with isoforms 1-4. This may explain the banding pattern observed here. Isoforms 2 and 3 are 115-kDa. Isoforms 1 and 4 are 116- and 113-kDa respectively. The band around 80-kDa corresponds to the predicted MW of isoform 6. However, ab93628 is not predicted to react with this isoform. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.