p53 Monoclonal Antibody (PAb 122) Catalog Number MA Product data sheet

Transcription

1 Website: thermofisher.com Customer Service (US): ext. 1 Technical Support (US): ext. 441 p53 (PAb 122) Catalog Number MA Product data sheet Details Size 500 µl Host/Isotope Mouse / IgG2b, kappa Class Monoclonal Type Antibody Clone PAb 122 Immunogen SV40-transformed mouse B4 cells Conjugate Unconjugated Form Liquid Concentration 0.2 mg/ml Purification Protein A Storage buffer PBS, ph 7.4, with 0.2% BSA Contains 0.09% sodium azide Storage Conditions 4 C Species Reactivity Tested species reactivity Published species reactivity Tested Applications Dilution * Immunofluorescence (IF) Immunohistochemistry (Paraffin) (IHC (P)) Immunoprecipitation (IP) Hamster, Human, Mouse, Nonhuman primate, Rat Human, Mouse, Not Applicable Assay Dependent 1:20 2 µg/ml Western Blot (WB) 1:500 Published Applications Western Blot (WB) Immunoprecipitation (IP) Immunocytochemistry (ICC) See 3 publications below See 3 publications below See 1 publications below * Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls. Product specific information MA targets p53 in FACS, IF, IP, and WB applications and shows reactivity with Hamster, Human, mouse, Non-human primate, and Rat samples. The MA immunogen is sv40-transformed mouse B4 cells. Background/Target Information This gene encodes a tumor suppressor protein containing transcriptional activation, DNA binding, and oligomerization domains. The encoded protein responds to diverse cellular stresses to regulate expression of target genes, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. Mutations in this gene are associated with a variety of human cancers, including hereditary cancers such as Li-Fraumeni syndrome. Alternative splicing of this gene and the use of alternate promoters result in multiple transcript variants and isoforms. Additional isoforms have also been shown to result from the use of alternate translation initiation codons

2 Advanced Verification Data p53 Antibody (MA ) in IP-MS IP-MS enrichment of TP53 (LFQ intensity): TP53 was enriched 3083-fold from BT549 lysate compared to background proteins, using the optimized IP-MS workflow with Pierce MS-Compatible Magnetic IP Kit protein A/G (Product # 90409) and TP53 antibody 12453). STRING database was used to identify the protein interactor list. See more information on IP-MS verification of antibody selectivity. IP-MS validation info. Product Images For p53 (PAb 122)

3 12453) in IHC (P) Immunohistochemistry analysis of p53 showing staining in the nucleus and weak staining in the cytoplasm of paraffinembedded human breast carcinoma tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was 10mM sodium citrate (ph 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddh2o and PBS, and then probed with a p53 Mouse 12453) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

4 12453) in IHC (P) Immunohistochemistry analysis of p53 showing staining in the nucleus and weak staining in the cytoplasm of paraffinembedded human colon carcinoma tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was 10mM sodium citrate (ph 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddh2o and PBS, and then probed with a p53 Mouse 12453) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

5 12453) in IHC (P) Immunohistochemistry analysis of p53 showing staining in the nucleus and weak staining in the cytoplasm of paraffinembedded mouse colon tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was 10mM sodium citrate (ph 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddh2o and PBS, and then probed with a p53 Mouse 12453) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

6 12453) in WB Western blot analysis was performed on whole cell extracts (30 µg lysate) of HEL (Lane 1), MDA- MB-231 (Lane 2), COS-7 (Lane 3), A- 431 (Lane 4), and SKOV3 (Lane 5). The blots were probed with Anti-p53 Mouse 12453, 1-2 µg/ml) and detected by chemiluminescence using Goat anti-mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # , 1:4000 dilution). A 53 kda band corresponding to p53 was observed across cell lines tested except SKOV3. An extra band at 100 kda also observed. Known quantity of protein samples were electrophoresed using Novex NuPAGE 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock Electrophoresis System (Product # EI0002) and Novex Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iblot 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was Novex ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). p53 Antibody (MA ) in WB

7 Western blot analysis of p53 was performed by loading 20 µg of MCF-7 (lane 1) and MDA-MB-231 (lane 2) cell lysates in RIPA buffer (Product # 89901) and Page Ruler Plus Protein Ladder (Product # 26620) onto a 4-8% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). Proteins were transferred to nitrocellulose membrane blocked in 5% milk /PBST for one hour at room temperature. p53 was detected using a cytochrome C monoclonal antibody 12453) at a dilution of 1:500 5% milk /PBST overnight at at 4 C on a rocking platform, followed by a Goat anti- Mouse IgG (H + L) - HRP secondary antibody at a dilution of 1:5,000 for one hour. Chemiluminescent detection was SuperSignal West Dura (Product # 34078) and Kodak M35 X-OMAT Automatic Processor. Data courtesy of Dr. Wei Xu at the University of Wisconsin, Madison, WI.

8 12453) in IP Immunoprecipitation of p53 using p ) on denatured Human MDA231 Cells.

9 PubMed References For p53 (PAb 122) 3 Western Blot References Species / Dilution Summary MA was used in immunocytochemistry and western blot to elucidate an increase of apoptosis and disruption of cytoskeleton organization of rat neural crest stem cells via upregulating CXCR4 expression and RhoA-ROCK1-p38 MAPKp53 signaling due to sim Not Applicable / Not Cited Human / Not Cited Stem cells and development (Aug 2016; 25: 1172) "Simulated Microgravity Disrupts Cytoskeleton Organization and Increases Apoptosis of Rat Neural Crest Stem Cells Via Upregulating CXCR4 Expression and RhoA-ROCK1-p38 MAPK-p53 Signaling." Author(s):Lin SC,Gou GH,Hsia CW,Ho CW,Huang KL,Wu YF,Lee SY,Chen YH PubMed Article URL: MA was used in western blot to study keratin-14 downregulation during epidermal cell differentiation and the corepressor role of p53 PloS one (Aug 2012; 7: null) "p53 acts as a co-repressor to regulate keratin 14 expression during epidermal cell differentiation." Author(s):Cai BH,Hsu PC,Hsin IL,Chao CF,Lu MH,Lin HC,Chiou SH,Tao PL,Chen JY PubMed Article URL: MA was used in western blot to study a novel p53-binding domain in CUL7 Mouse / Not Cited 3 Immunoprecipitation References Species / Dilution Biochemical and biophysical research communications (Sep 2006; 348: 132) "A novel p53-binding domain in CUL7." Author(s):Kasper JS,Arai T,DeCaprio JA PubMed Article URL: Summary MA was used in immunoprecipitation to study the role of p53 in the interaction between simian virus 40 large T antigen and human CBP/p300 Human / Not Cited Mouse / Not Cited Journal of virology (May 2006; 80: 4292) "Targeting of p300/creb binding protein coactivators by simian virus 40 is mediated through p53." Author(s):Borger DR,DeCaprio JA PubMed Article URL: MA was used in immunoprecipitation to study the expression of genes involved in cell cycle checkpoint activation and apoptosis in p53 heterozygous mice following X-ray irradiation Mutation research (Feb 2006; 594: 49) "Gene expression and apoptosis induction in p53-heterozygous irradiated mice." Author(s):di Masi A,Antoccia A,Dimauro I,Argentino-Storino A,Mosiello A,Mango R,Novelli G,Tanzarella C PubMed Article URL: MA was used in immunoprecipitation to study whether the loss of both p53 tumor suppressor alleles is required for tumor progression Mouse / Not Cited 1 Immunocytochemistry References Species / Dilution The EMBO journal (Aug 1998; 17: 4657) "Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation." Author(s):Venkatachalam S,Shi YP,Jones SN,Vogel H,Bradley A,Pinkel D,Donehower LA PubMed Article URL: Summary MA was used in immunocytochemistry and immunoprecipitation to study the mechanism by which p53 targets Delta Np63 into a protein degradation pathway Human / 1:100 Proceedings of the National Academy of Sciences of the United States of America (Feb 2001; 98: 1817) "p53 associates with and targets Delta Np63 into a protein degradation pathway." Author(s):Ratovitski EA,Patturajan M,Hibi K,Trink B,Yamaguchi K,Sidransky D PubMed Article URL: