p53 Total + pSer392 Human In-Cell ELISA Kit (IR) (ab128571)
- Product namep53 Total + pSer392 Human In-Cell ELISA Kit (IR)
- Detection methodIR
- Tests1 x 96 well plate
- Sample typeAdherent cells, Suspension cells
- Assay typeCell-based (quantitative)
- Assay durationMultiple steps standard assay
- Species reactivityReacts with: Human
- Product overview
p53 (TP53 gene) acts as a tumor suppressor in many tumor types and induces growth arrest or apoptosis depending on the physiological circumstances and cell type. p53 is involved in cell cycle regulation as a trans-activator that acts to negatively regulate cell division by controlling a set of genes required for this process. One of the activated genes is an inhibitor of cyclin-dependent kinases. p53 mediated apoptosis induction seems to be by stimulation of BAX and FAS antigen expression, or by repression of Bcl-2 expression. p53 is also implicated in Notch signaling cross-over.
In-Cell ELISA (ICE) technology is used to perform quantitative immunocytochemistry of cultured cells with a near-infrared fluorescent dye-labeled detector antibody. The technique generates quantitative data with specificity similar to Western blotting, but with much greater quantitative precision and higher throughput due to the greater dynamic range and linearity of direct fluorescence detection and the ability to run 96 samples in parallel. Because the p53 antibody is a mouse antibody and the p53 phospho S392 antibody is a rabbit antibody, their targets can be measured simultaneously in a same well using the cocktail of provided primary antibodies and the provided cocktail of IRDye®-labeled species-specific secondary antibodies when using a LI-COR infrared imager. This method rapidly fixes the cells in situ, stabilizing the in vivo levels of proteins and their post-translational modifications, and thus essentially eliminates changes during sample handling, such as preparation of protein extracts. Finally, the p53 phospho Ser392 and total p53 protein signals can all be normalized to cell amount, measured by the provided Janus Green whole cell stain, to further increase the assay precision.
Plates are available in our ICE (In-Cell ELISA) Support Pack (ab111542) which can be bought seperately.
- Tested applicationsIn-Cell ELISA more details
- Storage instructionsStore at +4°C. Please refer to protocols.
Components 1 x 96 tests 1000X IRDye® Labeled Secondary Antibody Cocktail 1 x 24µl 100X Mouse Anti-p53 Primary Antibody 1 x 0.12ml 100X Rabbit Anti-p53 Phospho S392 Primary Antibody 1 x 0.12ml 100X Triton X-100 1 x 0.5ml 10X Blocking Solution 1 x 5ml 10X Phosphate Buffered Saline 1 x 100ml 1X Janus Green Stain 1 x 11ml 400X Tween-20 1 x 2ml Incubation Buffer 1 x 60ml
- RelevanceThis gene encodes tumor protein p53, which responds to diverse cellular stresses to regulate target genes that induce cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. p53 protein is expressed at low level in normal cells and at a high level in a variety of transformed cell lines, where it's believed to contribute to transformation and malignancy. p53 is a DNA-binding protein containing transcription activation, DNA-binding, and oligomerization domains. It is postulated to bind to a p53-binding site and activate expression of downstream genes that inhibit growth and/or invasion, and thus function as a tumor suppressor. Mutants of p53 that frequently occur in a number of different human cancers fail to bind the consensus DNA binding site, and hence cause the loss of tumor suppressor activity. Alterations of this gene occur not only as somatic mutations in human malignancies, but also as germline mutations in some cancer-prone families with Li-Fraumeni syndrome. Multiple p53 variants due to alternative promoters and multiple alternative splicing have been found. These variants encode distinct isoforms, which can regulate p53 transcriptional activity.
- Cellular localizationCytoplasm. Nucleus. Nucleus › PML body. Endoplasmic reticulum. Note: Interaction with BANP promotes nuclear localization. Recruited into PML bodies together with CHEK2.
- Antigen NY-CO-1
- Cellular tumor antigen p53
- Phosphoprotein p53
- Transformation-related protein 53
- Tumor protein p53
- Tumor suppressor p53
Our Abpromise guarantee covers the use of ab128571 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|In-Cell ELISA||In-Cell ELISA|
p53 Total + pSer392 Human In-Cell ELISA Kit (IR) images
Sample experiment using ab128571 on MCF-7 cells treated with for 6 hours with Camptothecin or drug vehicle (DMSO). The data (presented as mean +/- SEM) were analyzed as described in Data Analysis section of the protocol.
Specificity of antibodies demonstrated by immuno-cytochemistry. Primary antibodies used in this assay kit were validated by staining MCF-7 cells treated with Camptothecin or vehicle and imaged by fluorescent microscopy. (1) Mostly nuclear localization of both p53 phospho S392 and total p53 protein signals, and (2) an increase in both p53 phospho S392 and total p53 protein signals upon Camptothecin treatment.
Antibody specificity demonstrated by Western Blot Analysis. Primary antibodies used in this assay kit were validated by Western Blotting using Hek293T induced with Etoposide (to increase the levels of p53 phosphorylation). Cell extract was treated with increasing concentrations of λ protein phosphatase (lane 2, 400x diluted; lane 3, 100x diluted; lane 4, 25x diluted) or left untreated (lane 1). Samples were analyzed by Western blotting using the rabbit p53 phospho S392 antibody (A). The membrane was re-probed with the mouse total p53 protein antibody (B).
References for p53 Total + pSer392 Human In-Cell ELISA Kit (IR) (ab128571)
ab128571 has not yet been referenced specifically in any publications.