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Typical phospho-p53 (Ser15) Standard Curve
100 μL of progressive 2 fold dilutions of the p53 standard included in the kit and run as des...
Phospho-p53 (Ser15) STAR ELISA Kit
Description:
Phospho-p53 (Ser15) STAR ELISA Kit
Trade Name:
Upstate (Millipore)
Qty/Pk:
96 assays
Background Information:
I. TEST PRINCIPLE
The colorimetric STAR (Signal Transduction Assay Reaction) ELISA kit is a solid phase sandwich enzyme linked immunosorbent assay that provides a fast, sensitive method to detect specific levels of signaling targets in whole cell extracts. The p53 plate is coated with a specific mouse monoclonal p53 capture antibody on the microwells of the 96-well clear plate. Sample lysate or the standard included in the kit are incubated in the microwells allowing p53 antigen to be captured in the plate wells. The plate is then washed to remove any unbound non-specific material. The wells are then incubated with a specific rabbit anti-phospho-p53 (Ser15) antibody to detect the captured p53 on the plate well that is phosphorylated on Ser15. The unbound detection antibody is washed away followed by incubation with an HRP-conjugated anti-rabbit antibody. After the addition of TMB substrate and stop solution the absorbance is measured at 450 nm using a plate reader. This allows for a sensitive enzymatic detection of the sample.
The entire assay takes less than 5 hours to complete with minimal hands-on time. Many of the reagents are supplied in ready-to use formulations for ease of use. The kit also includes a standard that is run as both a positive control and to generate a standard curve for phosphorylated p53 (Ser15) measurement.
II. p53 BACKGROUND
p53, a gene that is mutated in approximately half of all human cancers, has long been regarded as the guardian of the genome by regulating genes controlling cell cycle progression, DNA repair and apoptosis. In response to cellular stress, such as DNA damage, hypoxia, or radiation, p53 is activated and in turn it transcriptionally activates the expression of particular genes, including the cyclin-dependent kinase inhibitor p21, and together with p19ARF, induces expression of p21Cip1, to cause cell cycle arrest. Alternatively, p53 can work via apoptosis as a way of eliminating irreparably damaged cells.
Inactivation or loss of p53 has been associated with deregulation of the cell cycle and DNA replication, inefficient DNA repair, loss of cellular apoptotic responses and ultimately the development of various human cancers. The p53 polypeptide contains three distinct regions. The amino-terminal 83 amino acids of p53 contains the transactivation domain, as well as the region involved in transcription-independent growth suppression, the central sequence-specific DNA binding region, and the carboxy-terminal region contains the DNA-binding domain. Activation and stabilization of p53 is regulated by phosphorylation and possibly acetylation
The colorimetric STAR (Signal Transduction Assay Reaction) ELISA kit is a solid phase sandwich enzyme linked immunosorbent assay that provides a fast, sensitive method to detect specific levels of signaling targets in whole cell extracts. The p53 plate is coated with a specific mouse monoclonal p53 capture antibody on the microwells of the 96-well clear plate. Sample lysate or the standard included in the kit are incubated in the microwells allowing p53 antigen to be captured in the plate wells. The plate is then washed to remove any unbound non-specific material. The wells are then incubated with a specific rabbit anti-phospho-p53 (Ser15) antibody to detect the captured p53 on the plate well that is phosphorylated on Ser15. The unbound detection antibody is washed away followed by incubation with an HRP-conjugated anti-rabbit antibody. After the addition of TMB substrate and stop solution the absorbance is measured at 450 nm using a plate reader. This allows for a sensitive enzymatic detection of the sample.
The entire assay takes less than 5 hours to complete with minimal hands-on time. Many of the reagents are supplied in ready-to use formulations for ease of use. The kit also includes a standard that is run as both a positive control and to generate a standard curve for phosphorylated p53 (Ser15) measurement.
II. p53 BACKGROUND
p53, a gene that is mutated in approximately half of all human cancers, has long been regarded as the guardian of the genome by regulating genes controlling cell cycle progression, DNA repair and apoptosis. In response to cellular stress, such as DNA damage, hypoxia, or radiation, p53 is activated and in turn it transcriptionally activates the expression of particular genes, including the cyclin-dependent kinase inhibitor p21, and together with p19ARF, induces expression of p21Cip1, to cause cell cycle arrest. Alternatively, p53 can work via apoptosis as a way of eliminating irreparably damaged cells.
Inactivation or loss of p53 has been associated with deregulation of the cell cycle and DNA replication, inefficient DNA repair, loss of cellular apoptotic responses and ultimately the development of various human cancers. The p53 polypeptide contains three distinct regions. The amino-terminal 83 amino acids of p53 contains the transactivation domain, as well as the region involved in transcription-independent growth suppression, the central sequence-specific DNA binding region, and the carboxy-terminal region contains the DNA-binding domain. Activation and stabilization of p53 is regulated by phosphorylation and possibly acetylation
Key Applications:
ELISA
Species Reactivity:
Human
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Entrez Gene Summary:
Tumor protein p53, a nuclear protein, plays an essential role in the regulation of cell cycle, specifically in the transition from G0 to G1. It is found in very low levels in normal cells, however, in a variety of transformed cell lines, it is expressed in high amounts, and believed to contribute to transformation and malignancy. p53 is a DNA-binding protein containing DNA-binding, oligomerization and transcription activation domains. It is postulated to bind as a tetramer 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 the TP53 gene occur not only as somatic mutations in human malignancies, but also as germline mutations in some cancer-prone families with Li-Fraumeni syndrome.
UniProt Summary:
FUNCTION: SwissProt: P04637 # Acts as a tumor suppressor in many tumor types; induces growth arrest or apoptosis depending on the physiological circumstances and cell type. 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. Apoptosis induction seems to be mediated either by stimulation of BAX and FAS antigen expression, or by repression of Bcl-2 expression.
COFACTOR: Binds 1 zinc ion per subunit.
SIZE: 393 amino acids; 43653 Da
SUBUNIT: Interacts with AXIN1. Probably part of a complex consisting of TP53, HIPK2 and AXIN1 (By similarity). Binds DNA as a homotetramer. Interacts with histone acetyltransferases EP300 and methyltransferases HRMT1L2 and CARM1, and recruits them to promoters. In vitro, the interaction of TP53 with cancer- associated/HPV (E6) viral proteins leads to ubiquitination and degradation of TP53 giving a possible model for cell growth regulation. This complex formation requires an additional factor, E6-AP, which stably associates with TP53 in the presence of E6. C- terminus interacts with TAF1, when TAF1 is part of the TFIID complex. Interacts with ING4 and this interaction may be indirect. Found in a complex with CABLES1 and TP73. Interacts with HIPK1, HIPK2, and P53DINP1. Interacts with WWOX. May interacts with HCV core protein. Interacts with USP7 and SYVN1. Interacts with HSP90AB1 (By similarity). Interacts with BANP.
SUBCELLULAR LOCATION: Cytoplasm. Nucleus. Endoplasmic reticulum. Note=Interaction with BANP promotes nuclear localization.
DOMAIN: SwissProt: P04637 The nuclear export signal acts as a transcriptional repression domain.
PTM: Acetylated. Acetylation of Lys-382 by CREBBP enhances transcriptional activity. Deacetylation of Lys-382 by SIRT1 impairs its ability to induce proapoptotic program and modulate cell senescence. & Phosphorylation on Ser residues mediates transcriptional activation. Phosphorylated by HIPK1 (By similarity). Phosphorylated on Thr-18 by VRK1, which may prevent the interaction with MDM2. Phosphorylated on Thr-55 by TAF1, which promotes MDM2-mediated degradation. Phosphorylated on Ser-46 by HIPK2 upon UV irradiation. Phosphorylation on Ser-46 is required for acetylation by CREBBP. Phosphorylated on Ser-392 following UV but not gamma irradiation. Phosphorylated upon DNA damage, probably by ATM or ATR. Phosphorylated on Ser-15 upon ultraviolet irradiation; which is enhanced by interaction with BANP. & Dephosphorylated by PP2A. SV40 small T antigen inhibits the dephosphorylation by the AC form of PP2A. & May be O-glycosylated in the C-terminal basic region. Studied in EB-1 cell line. & Ubiquitinated by SYVN1, which leads to proteasomal degradation.
DISEASE: SwissProt: P04637 # TP53 is found in increased amounts in a wide variety of transformed cells. TP53 is frequently mutated or inactivated in about 60% of cancers. & Defects in TP53 are involved in esophageal squamous cell carcinoma (ESCC) [MIM:133239]. ESCC is a tumor of the esophagus. & Defects in TP53 are a cause of Li-Fraumeni syndrome (LFS) [MIM:151623]. LFS is an autosomal dominant familial cancer syndrome that in its classic form is defined by the existence of both a proband with a sarcoma and two other first-degree relatives with a cancer by age 45 years. In these families the affected relatives develop a diverse set of malignancies at unusually early ages. The spectrum of cancers in LFS includes breast carcinomas, soft-tissue sarcomas, brain tumors, osteosarcoma, leukemia and adreno-cortical carcinoma. Other possible component tumors of LFS are melanoma, gonadal cell tumors and carcinomas of the lung, pancreas and prostate. & Defects in TP53 may be associated with nasopharyngeal carcinoma [MIM:161550]; also known as nasopharyngeal cancer. & Defects in TP53 are found in Barrett metaplasia; also known as Barrett esophagus. It is a condition in which the normally stratified squamous epithelium of the lower esophagus is replaced by a metaplastic columnar epithelium. The condition develops as a complication in approximately 10% of patients with chronic gastroesophageal reflux disease and predisposes to the development of esophageal adenocarcinoma. & Defects in TP53 are involved in head and neck squamous cell carcinomas (HNSCC) [MIM:275355]. & Defects in TP53 are involved in oral squamous cell carcinoma (OSCC). Cigarette smoke is a prime mutagenic agent in cancer of the aerodigestive tract. & Defects in TP53 are a cause of lung cancer [MIM:211980]. & Defects in TP53 are a cause of choroid plexus papilloma [MIM:260500]. Choroid plexus papilloma is a slow-growing benign tumor of the choroid plexus that often invades the leptomeninges. In children it is usually in a lateral ventricle but in adults it is more often in the fourth ventricle. Hydrocephalus is common, either from obstruction or from tumor secretion of cerebrospinal fluid. If it undergoes malignant transformation it is called a choroid plexus carcinoma. Primary choroid plexus tumors are rare and usually occur in early childhood. & Defects in TP53 are a cause of one form of hereditary adrenocortical carcinoma (ADCC) [MIM:202300]. ADCC is a rare childhood tumor, representing about 0.4% of childhood tumors, with a high incidence of associated tumors. ADCC occurs with increased frequency in patients with the Beckwith-Wiedemann syndrome [MIM:130650] and is a component tumor in Li-Fraumeni syndrome [MIM:151623].
SIMILARITY: Belongs to the p53 family.
COFACTOR: Binds 1 zinc ion per subunit.
SIZE: 393 amino acids; 43653 Da
SUBUNIT: Interacts with AXIN1. Probably part of a complex consisting of TP53, HIPK2 and AXIN1 (By similarity). Binds DNA as a homotetramer. Interacts with histone acetyltransferases EP300 and methyltransferases HRMT1L2 and CARM1, and recruits them to promoters. In vitro, the interaction of TP53 with cancer- associated/HPV (E6) viral proteins leads to ubiquitination and degradation of TP53 giving a possible model for cell growth regulation. This complex formation requires an additional factor, E6-AP, which stably associates with TP53 in the presence of E6. C- terminus interacts with TAF1, when TAF1 is part of the TFIID complex. Interacts with ING4 and this interaction may be indirect. Found in a complex with CABLES1 and TP73. Interacts with HIPK1, HIPK2, and P53DINP1. Interacts with WWOX. May interacts with HCV core protein. Interacts with USP7 and SYVN1. Interacts with HSP90AB1 (By similarity). Interacts with BANP.
SUBCELLULAR LOCATION: Cytoplasm. Nucleus. Endoplasmic reticulum. Note=Interaction with BANP promotes nuclear localization.
DOMAIN: SwissProt: P04637 The nuclear export signal acts as a transcriptional repression domain.
PTM: Acetylated. Acetylation of Lys-382 by CREBBP enhances transcriptional activity. Deacetylation of Lys-382 by SIRT1 impairs its ability to induce proapoptotic program and modulate cell senescence. & Phosphorylation on Ser residues mediates transcriptional activation. Phosphorylated by HIPK1 (By similarity). Phosphorylated on Thr-18 by VRK1, which may prevent the interaction with MDM2. Phosphorylated on Thr-55 by TAF1, which promotes MDM2-mediated degradation. Phosphorylated on Ser-46 by HIPK2 upon UV irradiation. Phosphorylation on Ser-46 is required for acetylation by CREBBP. Phosphorylated on Ser-392 following UV but not gamma irradiation. Phosphorylated upon DNA damage, probably by ATM or ATR. Phosphorylated on Ser-15 upon ultraviolet irradiation; which is enhanced by interaction with BANP. & Dephosphorylated by PP2A. SV40 small T antigen inhibits the dephosphorylation by the AC form of PP2A. & May be O-glycosylated in the C-terminal basic region. Studied in EB-1 cell line. & Ubiquitinated by SYVN1, which leads to proteasomal degradation.
DISEASE: SwissProt: P04637 # TP53 is found in increased amounts in a wide variety of transformed cells. TP53 is frequently mutated or inactivated in about 60% of cancers. & Defects in TP53 are involved in esophageal squamous cell carcinoma (ESCC) [MIM:133239]. ESCC is a tumor of the esophagus. & Defects in TP53 are a cause of Li-Fraumeni syndrome (LFS) [MIM:151623]. LFS is an autosomal dominant familial cancer syndrome that in its classic form is defined by the existence of both a proband with a sarcoma and two other first-degree relatives with a cancer by age 45 years. In these families the affected relatives develop a diverse set of malignancies at unusually early ages. The spectrum of cancers in LFS includes breast carcinomas, soft-tissue sarcomas, brain tumors, osteosarcoma, leukemia and adreno-cortical carcinoma. Other possible component tumors of LFS are melanoma, gonadal cell tumors and carcinomas of the lung, pancreas and prostate. & Defects in TP53 may be associated with nasopharyngeal carcinoma [MIM:161550]; also known as nasopharyngeal cancer. & Defects in TP53 are found in Barrett metaplasia; also known as Barrett esophagus. It is a condition in which the normally stratified squamous epithelium of the lower esophagus is replaced by a metaplastic columnar epithelium. The condition develops as a complication in approximately 10% of patients with chronic gastroesophageal reflux disease and predisposes to the development of esophageal adenocarcinoma. & Defects in TP53 are involved in head and neck squamous cell carcinomas (HNSCC) [MIM:275355]. & Defects in TP53 are involved in oral squamous cell carcinoma (OSCC). Cigarette smoke is a prime mutagenic agent in cancer of the aerodigestive tract. & Defects in TP53 are a cause of lung cancer [MIM:211980]. & Defects in TP53 are a cause of choroid plexus papilloma [MIM:260500]. Choroid plexus papilloma is a slow-growing benign tumor of the choroid plexus that often invades the leptomeninges. In children it is usually in a lateral ventricle but in adults it is more often in the fourth ventricle. Hydrocephalus is common, either from obstruction or from tumor secretion of cerebrospinal fluid. If it undergoes malignant transformation it is called a choroid plexus carcinoma. Primary choroid plexus tumors are rare and usually occur in early childhood. & Defects in TP53 are a cause of one form of hereditary adrenocortical carcinoma (ADCC) [MIM:202300]. ADCC is a rare childhood tumor, representing about 0.4% of childhood tumors, with a high incidence of associated tumors. ADCC occurs with increased frequency in patients with the Beckwith-Wiedemann syndrome [MIM:130650] and is a component tumor in Li-Fraumeni syndrome [MIM:151623].
SIMILARITY: Belongs to the p53 family.
Components:
- Capture Plate pre-coated with anti-p53 antibody: (Part No. 17-475A) One pre-coated 96-stripwell immunoplate sealed in a foil pouch.
- Anti-phospho-p53 (Ser15) detection antibody: (Part No. 17-475B) One bottle (11 mL) of anti-phospho-p53 (Ser15) detection antibody containing sodium azide, ready to use.
- ELISA Diluent: (Part No. 17-475C) One bottle (25 mL) of ELISA Diluent containing sodium azide, ready to use.
- 25X ELISA Wash Buffer: (Part No. 17-475D) One bottle (50 mL) of 25X ELISA Wash Buffer.
- Anti-Rabbit IgG HRP conjugate: (Part No. 17-475E) One vial (125 μL) of 100X anti-rabbit HRP conjugate
- HRP Diluent: (Part No. 17-475F) One bottle (25 mL) of HRP Diluent.
- TMB Solution: (Part No. 17-475G) One bottle (25 mL) of stabilized tetramethylbenzidine (TMB), ready to use.
- Stop Solution: (Part No. 17-475H) One bottle (25 mL) of stop solution, ready to use.
- Phospho-p53 (Ser15) Standard: (Part No. 17-475I) Two vials of phosphorylated p53 (Ser15) standard, lyophilized.
- Plate Covers: Two plate covers
Brand Family:
Upstate
Sensitivity:
Sensitivity: 1 units/mL
Range of Detection: 1.6 to 100 units/mL
Range of Detection: 1.6 to 100 units/mL
UniProt Number:
Storage Conditions:
Maintain the unopened kit at 2-8°C until expiration date.
Precautions
• The instructions provided have been designed to optimize the kit's performance. Deviation from the instructions may result in suboptimal performance of the kit and the failure to produce accurate data.
• Caustic Material: Stop Solution. Caution: Eye, hand, face, and clothing protection should be worn when handling this material.
• Safety Warnings and Precautions: This kit is designed for research use only and not recommended for internal use in humans or animals. All chemicals should be considered potentially hazardous and principles of good laboratory practice should be followed.
• The Detection Antibody and ELISA Diluent contain sodium azide. Sodium azide may react with copper and lead plumbing to form highly explosive metal azides. Upon disposal, flush with large amounts of water to prevent azide build-up. Avoid contact with skin.
• The Anti-Rabbit IgG HRP Conjugate and HRP Diluent contain thimerosal. Thimerosal is highly toxic by inhalation, contact with skin or if swallowed. Thimerosal is a possible mutagen and should be handled accordingly.
Precautions
• The instructions provided have been designed to optimize the kit's performance. Deviation from the instructions may result in suboptimal performance of the kit and the failure to produce accurate data.
• Caustic Material: Stop Solution. Caution: Eye, hand, face, and clothing protection should be worn when handling this material.
• Safety Warnings and Precautions: This kit is designed for research use only and not recommended for internal use in humans or animals. All chemicals should be considered potentially hazardous and principles of good laboratory practice should be followed.
• The Detection Antibody and ELISA Diluent contain sodium azide. Sodium azide may react with copper and lead plumbing to form highly explosive metal azides. Upon disposal, flush with large amounts of water to prevent azide build-up. Avoid contact with skin.
• The Anti-Rabbit IgG HRP Conjugate and HRP Diluent contain thimerosal. Thimerosal is highly toxic by inhalation, contact with skin or if swallowed. Thimerosal is a possible mutagen and should be handled accordingly.
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Detection Methods:
Colorimetric
Modifications:
Phosphorylation
Gene Symbol:
- TP53
- P53
- TRP53
- p53
- LFS1
Product Name:
Phospho-p53 (Ser15) STAR ELISA Kit
Entrez Gene Number:
Materials Required but Not Delivered:
1. Multi-channel or repeating pipettes
2. Plate shaker (optional)
3. Pipettors and tips capable of accurately measuring 1-1000 µL
4. Graduated serological pipettes
5. 96-well microtiter Plate Reader with 450 nm filter
6. Graphing software for plotting data or graph paper for manual plotting of data
7. Microfuge tubes for standard and sample dilutions
8. Mechanical vortex
9. 1 liter container
10. Distilled or deionized water
2. Plate shaker (optional)
3. Pipettors and tips capable of accurately measuring 1-1000 µL
4. Graduated serological pipettes
5. 96-well microtiter Plate Reader with 450 nm filter
6. Graphing software for plotting data or graph paper for manual plotting of data
7. Microfuge tubes for standard and sample dilutions
8. Mechanical vortex
9. 1 liter container
10. Distilled or deionized water