Anti-p53 抗体 [DO-1] - ChIP Grade (ab1101)
Key features and details
- Mouse monoclonal [DO-1] to p53 - ChIP Grade
- Suitable for: ChIP, IHC-P, Flow Cyt (Intra), WB, ICC/IF
- Knockout validated
- Reacts with: Human
- Isotype: IgG2a
Related conjugates and formulations
製品の概要
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製品名
Anti-p53 antibody [DO-1] - ChIP Grade
p53 一次抗体 製品一覧 -
製品の詳細
Mouse monoclonal [DO-1] to p53 - ChIP Grade -
由来種
Mouse -
特異性
This antibody clone recognises both wild-type and mutant forms of p53 in human samples. It is not designed to recognise any specific p53 mutation.
We have confirmed this experimentally and have been able to detect p53 in different cell lines using various applications and treatments.
Important note: p53 expression levels vary greatly between cell lines. It has been reported that p53 mutations render the protein more stable, hence mutated cell lines often express higher levels of the p53 protein compared to wild-type cell lines. For low expressing wild type cell lines, p53 expression can be increased with cell treatments such as camptothecin or irinotecan.
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アプリケーション
適用あり: ChIP, IHC-P, Flow Cyt (Intra), WB, ICC/IFmore details -
種交差性
交差種: Human
非交差種: Mouse, Rat -
免疫原
Recombinant full length protein. This information is proprietary to Abcam and/or its suppliers.
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エピトープ
The epitope maps to within aa 20-25. -
ポジティブ・コントロール
- Wild type p53: A549, HEK293, HepG2, MCF7, U-87 MG. Mutant p53: A431 (R273H), DU 145 (P223L and V274F), HAP1 (S215G), Jurkat (R196*), MDA-MB-435 (G266E), Raji (R213Q and Y234H), Ramos (I254N), SK-BR-3 (R175H), T-47D (L194F). Cell lines expressing the highest levels of p53 without induction are HEK293 (WT p53), A431 and HAP1 (mutant p53). Negative cell line: Saos-2. IHC-P controls: Bladder, Skin Cancer, Glioma, Gastric adenocarcinoma, Human breast and lung carcinoma tissue, Human colon adenocarcinoma.
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特記事項
This monoclonal p53 antibody has been knockout validated in Western blot. The expected band for p53 was observed in wild type HAP1 cell lysate and the band was not seen in TP53 knockout HAP1 cell lysate.
This antibody clone is manufactured by Abcam. If you require a custom buffer formulation or conjugation for your experiments, please contact orders@abcam.com.
The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.
If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As
製品の特性
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製品の状態
Liquid -
保存方法
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
バッファー
pH: 7.40
Preservative: 0.02% Sodium azide
Constituents: PBS, 6.97% L-Arginine -
Concentration information loading...
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精製度
Protein G purified -
ポリ/モノ
モノクローナル -
クローン名
DO-1 -
ミエローマ
unknown -
アイソタイプ
IgG2a -
軽鎖の種類
kappa -
研究分野
関連製品
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Alternative Versions
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Assay kits
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ChIP Related Products
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Compatible Secondaries
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Isotype control
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Positive Controls
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Recombinant Protein
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Related Products
アプリケーション
The Abpromise guarantee
Abpromise保証は、 次のテスト済みアプリケーションにおけるab1101の使用に適用されます
アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。
アプリケーション | Abreviews | 特記事項 |
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ChIP |
Use at an assay dependent concentration.
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IHC-P |
Use a concentration of 1 - 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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Flow Cyt (Intra) |
Use at an assay dependent concentration.
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WB | (8) |
Use at an assay dependent concentration. Predicted molecular weight: 43 kDa.
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ICC/IF | (2) |
Use at an assay dependent concentration.
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特記事項 |
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ChIP
Use at an assay dependent concentration. |
IHC-P
Use a concentration of 1 - 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
Flow Cyt (Intra)
Use at an assay dependent concentration. |
WB
Use at an assay dependent concentration. Predicted molecular weight: 43 kDa. |
ICC/IF
Use at an assay dependent concentration. |
ターゲット情報
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機能
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. Implicated in Notch signaling cross-over. Isoform 2 enhances the transactivation activity of isoform 1 from some but not all TP53-inducible promoters. Isoform 4 suppresses transactivation activity and impairs growth suppression mediated by isoform 1. Isoform 7 inhibits isoform 1-mediated apoptosis. -
組織特異性
Ubiquitous. Isoforms are expressed in a wide range of normal tissues but in a tissue-dependent manner. Isoform 2 is expressed in most normal tissues but is not detected in brain, lung, prostate, muscle, fetal brain, spinal cord and fetal liver. Isoform 3 is expressed in most normal tissues but is not detected in lung, spleen, testis, fetal brain, spinal cord and fetal liver. Isoform 7 is expressed in most normal tissues but is not detected in prostate, uterus, skeletal muscle and breast. Isoform 8 is detected only in colon, bone marrow, testis, fetal brain and intestine. Isoform 9 is expressed in most normal tissues but is not detected in brain, heart, lung, fetal liver, salivary gland, breast or intestine. -
関連疾患
Note=TP53 is found in increased amounts in a wide variety of transformed cells. TP53 is frequently mutated or inactivated in about 60% of cancers. TP53 defects are found in Barrett metaplasia 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 a cause of esophageal cancer (ESCR) [MIM:133239].
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 a proband affected by a sarcoma before 45 years with a first degree relative affected by any tumor before 45 years and another first degree relative with any tumor before 45 years or a sarcoma at any age. Other clinical definitions for LFS have been proposed (PubMed:8118819 and PubMed:8718514) and called Li-Fraumeni like syndrome (LFL). In these families affected relatives develop a diverse set of malignancies at unusually early ages. Four types of cancers account for 80% of tumors occurring in TP53 germline mutation carriers: breast cancers, soft tissue and bone sarcomas, brain tumors (astrocytomas) and adrenocortical carcinomas. Less frequent tumors include choroid plexus carcinoma or papilloma before the age of 15, rhabdomyosarcoma before the age of 5, leukemia, Wilms tumor, malignant phyllodes tumor, colorectal and gastric cancers.
Defects in TP53 are involved in head and neck squamous cell carcinomas (HNSCC) [MIM:275355]; also known as squamous cell carcinoma of the head and neck.
Defects in TP53 are a cause of lung cancer (LNCR) [MIM:211980].
Defects in TP53 are a cause of choroid plexus papilloma (CPLPA) [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 adrenocortical carcinoma (ADCC) [MIM:202300]. ADCC is a rare childhood tumor of the adrenal cortex. It occurs with increased frequency in patients with the Beckwith-Wiedemann syndrome and is a component tumor in Li-Fraumeni syndrome. -
配列類似性
Belongs to the p53 family. -
ドメイン
The nuclear export signal acts as a transcriptional repression domain. The TADI and TADII motifs (residues 17 to 25 and 48 to 56) correspond both to 9aaTAD motifs which are transactivation domains present in a large number of yeast and animal transcription factors. -
翻訳後修飾
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). Phosphorylation at Ser-9 by HIPK4 increases repression activity on BIRC5 promoter. Phosphorylated on Thr-18 by VRK1. Phosphorylated on Ser-20 by CHEK2 in response to DNA damage, which prevents ubiquitination by 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-PPP2R5C holoenzyme at Thr-55. 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 MDM2 and SYVN1, which leads to proteasomal degradation. Ubiquitinated by RFWD3, which works in cooperation with MDM2 and may catalyze the formation of short polyubiquitin chains on p53/TP53 that are not targeted to the proteasome. Ubiquitinated by MKRN1 at Lys-291 and Lys-292, which leads to proteasomal degradation. Deubiquitinated by USP10, leading to its stabilization. Ubiquitinated by TRIM24, which leads to proteasomal degradation. Ubiquitination by TOPORS induces degradation. Deubiquitination by USP7, leading to stabilization. Isoform 4 is monoubiquitinated in an MDM2-independent manner.
Monomethylated at Lys-372 by SETD7, leading to stabilization and increased transcriptional activation. Monomethylated at Lys-370 by SMYD2, leading to decreased DNA-binding activity and subsequent transcriptional regulation activity. Lys-372 monomethylation prevents interaction with SMYD2 and subsequent monomethylation at Lys-370. Dimethylated at Lys-373 by EHMT1 and EHMT2. Monomethylated at Lys-382 by SETD8, promoting interaction with L3MBTL1 and leading to repress transcriptional activity. Demethylation of dimethylated Lys-370 by KDM1A prevents interaction with TP53BP1 and represses TP53-mediated transcriptional activation.
Sumoylated by SUMO1. -
細胞内局在
Cytoplasm; Cytoplasm. Nucleus. Nucleus > PML body. Endoplasmic reticulum. Interaction with BANP promotes nuclear localization. Recruited into PML bodies together with CHEK2; Nucleus. Cytoplasm. Localized in both nucleus and cytoplasm in most cells. In some cells, forms foci in the nucleus that are different from nucleoli; Nucleus. Cytoplasm. Localized in the nucleus in most cells but found in the cytoplasm in some cells; Nucleus. Cytoplasm. Localized mainly in the nucleus with minor staining in the cytoplasm; Nucleus. Cytoplasm. Predominantly nuclear but localizes to the cytoplasm when expressed with isoform 4 and Nucleus. Cytoplasm. Predominantly nuclear but translocates to the cytoplasm following cell stress. - Information by UniProt
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参照データベース
- Entrez Gene: 7157 Human
- Omim: 191170 Human
- SwissProt: P04637 Human
- Unigene: 654481 Human
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別名
- Antigen NY-CO-13 antibody
- BCC7 antibody
- Cellular tumor antigen p53 antibody
see all
画像
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Flow cytometry overlay histogram showing mutant p53 in A-431 cells stained with ab1101 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab1101) (1x 106 cells in 100μl at 0.04μg/ml (1/25000)) for 30min at 22°C. The secondary antibody Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) was incubated at 1/4000 for 30min at 22°C. Isotype control antibody (black line) Mouse IgG2a, Kappa Monoclonal [MOPC-173] - Isotype Control - ChIP Grade (ab18413) was used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control. Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. This antibody gave a positive signal in A-431 fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.
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Flow cytometry overlay histogram showing wild-type p53 in Hek-293 positive cells (left) and MCF7 negative cells (right) stained with ab1101 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab1101) (1x 106 cells in 100μl at 0.2μg/ml (1/5000)) for 30min at 22°C. The secondary antibody Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) was incubated at 1/4000 for 30min at 22°C. Isotype control antibody (black line) Mouse IgG2a, Kappa Monoclonal [MOPC-173] - Isotype Control - ChIP Grade (ab18413) was used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control. Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. This antibody gave a positive signal in Hek-293 fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.
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Flow cytometry overlay histogram showing mutant p53 in wild-type HAP1 (green line) and TP53 knockout HAP1 cells (red line) stained with ab1101. The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab1101) (1x 106 cells in 100μl at 0.04 μg/ml (1/25000)) for 30min at 22°C.The secondary antibody Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) was incubated at 1/4000 for 30min at 22°C. Isotype control antibody Mouse IgG2a, Kappa Monoclonal [MOPC-173] - Isotype Control - ChIP Grade (ab18413) was used at the same concentration and conditions as the primary antibody (wild-type HAP1 - black line, TP53 knockout HAP1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. This antibody gave a positive signal in HAP1 fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.
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All lanes : Anti-p53 antibody [DO-1] - ChIP Grade (ab1101) at 1/1000 dilution
Lane 1 : MCF7 Treated Camptothecin (20 ug/ml, 0 h) cell lysate
Lane 2 : MCF7 Treated Camptothecin (20 ug/ml, 6 h) cell lysate
Lane 3 : MCF7 Treated Camptothecin (20 ug/ml, 24 h) cell lysate
Lane 4 : Wild-type A549 Treated Camptothecin (20 ug/ml, 0 h) cell lysate
Lane 5 : Wild-type A549 Treated Camptothecin (20 ug/ml, 6 h) cell lysate
Lane 6 : Wild-type A549 Treated Camptothecin (20 ug/ml, 24 h) cell lysate
Lane 7 : TP53 knockout A549 Treated Camptothecin (20 ug/ml, 0 h) cell lysate
Lane 8 : TP53 knockout A549 Treated Camptothecin (20 ug/ml, 6 h) cell lysate
Lane 9 : TP53 knockout A549 Treated Camptothecin (20 ug/ml, 24 h) cell lysate
Lane 10 : Saos-2 cell lysate
Lane 11 : SK-BR-3 cell lysate
Lane 12 : A431 cell lysate
Lysates/proteins at 15 µg per lane.
Performed under reducing conditions.
Predicted band size: 43 kDa
Observed band size: 40,50 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-p53 antibody [DO-1] - ChIP Grade staining at 1/1000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab1101 was shown to bind specifically to p53. A band was observed at 40/50 kDa in treated wild-type A549 cell lysates with no signal observed at this size in tp53 knockout cell line ab276092. To generate this image, wild-type and tp53 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.
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ab1101 staining mutant p53 in A431 cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab1101 at 0.2µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).
Also suitable in cells fixed with 100% methanol (5 min).
Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.
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ab1101 staining wild-type p53 in Hek293 cells (a high expressing cell line). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab1101 at 0.2µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).
Also suitable in cells fixed with 4% paraformaldehyde (10 min).
Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.
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ab1101 staining wild-type p53 in MCF7 cells (a low expressing cell line). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab1101 at 0.2µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).
Also suitable in cells fixed with 4% paraformaldehyde (10 min).
Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.
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All lanes : Anti-p53 antibody [DO-1] - ChIP Grade (ab1101) at 1/1000 dilution
Lane 1 : Saos-2 cell lysate
Lane 2 : A431 cell lysate
Lane 3 : Wild-type HAP1 cell lysate
Lane 4 : TP53 knockout HAP1 cell lysate
Lane 5 : MCF7 cell lysate
Lane 6 : HEK-293T cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 43 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-p53 antibody [DO-1] - ChIP Grade staining at 1/1000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab1101 was shown to bind specifically to p53. A band was observed at 50 kDa in wild-type HAP1 cell lysate with no signal observed at this size in tp53 knockout cell line. To generate this image, wild-type and tp53 knockout HAP1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.
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All lanes : Anti-p53 antibody [DO-1] - ChIP Grade (ab1101) at 1/1000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : TP53 knockout A549 cell lysate
Lane 3 : Wild-type Jurkat cell lysate
Lane 4 : TP53 knockout Jurkat cell lysate
Lane 5 : A431 cell lysate
Lane 6 : Saos-2 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 43 kDa
Observed band size: 49 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-p53 antibody [DO-1] - ChIP Grade staining at 1/1000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab1101 was shown to bind specifically to p53. A band was observed at 49 kDa in wild-type A549 and Jurkat cell lysates with no signal observed at this size in tp53 knockout cell lines ab276092, ab276112 (knockout cell lysates ab282999, ab283832). To generate this image, wild-type and tp53 knockout A549 and Jurkat cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.
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ab1101 staining p53 in wild-type Hap1 cells (top panel) and p53 knockout Hap1 cells (bottom panel). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab1101 at 1µg/ml concentration and ab6046 (Rabbit polyclonal to beta Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor® 594) (ab150080) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a high-content analysis system (Perkin Elmer, Operetta CLS™).
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Chromatin was prepared from HEK-293 (human epithelial cell line from embryonic kidney) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25 µg of chromatin, 2 µg of ab1101, and 10 ml of protein A sepharose beads,10 ml of protein G sepharose beads. No IgG was added to the beads control. The immunoprecipitated DNA was quantified by real time PCR. Primers were as follows:
Bax-1, forward: GGGTTATCTCTTGGGCTCACAA.
Bax-1, reverse: GAGCTCTCCCCAGCGCA.
Bax-2, forward: TGG AGC TGC AGA GGA TGA TTG
Bax-2, reverse: CCA GTT GAA GTT GCC GTC AGA
PUMA, forward: ATG CCT GCC TCA CCT TCA TC
PUMA, reverse: TCA CAC GTC GCT CTC TCT AAA CC
p21-1, forward: GCT GTG GCT CTG ATT GGC TTT
p21-1, reverse: ACA GGC AGC CCA AGG ACA AA
p21-2, forward: CAT CCC CAC AGC AGA GGA GAA
p21-2, reverse: ACC CAG GCT TGG AGC AGC TA
p21-3, forward: GAG TCC TGT TTG CTT CTG GGC A
p21-3, reverse: CTG CAT TGG GGC TGC CTA TGT A
PCNA, forward: CCA CCA TAA AGC TGG GGC TT
PCNA, reverse: TCT CCC CGC CTC TTT GAC TC
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All lanes : Anti-p53 antibody [DO-1] - ChIP Grade (ab1101) at 0.4 µg/ml
Lane 1 : Extract of HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells incubated with vehicle at 20 µg
Lane 2 : Extract of HEK-293T cells incubated with etoposide at 20 µg
Lane 3 : Extract of HEK-293T cells incubated with etoposide at 7.5 µg
Lane 4 : Lambda phosphatase (diluted 1/400)-treated extract of HEK-293T cells incubated with etoposide at 7.5 µg
Lane 5 : Lambda phosphatase (diluted 1/100)-treated extract of HEK-293T cells incubated with etoposide at 7.5 µg
Lane 6 : Lambda phosphatase (diluted 1/25)-treated extract of HEK-293T cells incubated with etoposide at 7.5 µg
Lane 7 : Extract of MCF7 (human breast adenocarcinoma cell line) cells incubated with vehicle at 20 µg
Lane 8 : Extract of MCF7 cells incubated 6 hours with camptothecin at 20 µg
Lane 9 : Extract of MCF7 cells incubated 16 hours with camptothecin at 20 µg
Lane 10 : Extract of MCF7 cells incubated 24 hours with camptothecin at 20 µg
Secondary
All lanes : Goat anti mouse IgG(H&L)-HRP at 1/10000 dilution
Predicted band size: 43 kDa
Observed band size: 53 kDa why is the actual band size different from the predicted?Developed using the ECL technique with 30 second exposure.
Blocking: 5% milk in PBS for 3 hours at RT.
Primary antibody in 5% BSA + PBS was incubated overnight at 4°C.
Secondary antibody in 5% milk + PBS was incubated for 2 hours at RT. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53 antibody [DO-1] - ChIP Grade (ab1101)
IHC image of ab1101 staining p53 in human colon adenocarcinoma formalin-fixed paraffin-embedded tissue sections*, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab1101, 1/500 dilution, for 15 minutes 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. High magnification of the tumor region - T (lower right panel) and adjacent normal crypts - N (lower left panel) are shown.
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.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre -
All lanes : Anti-p53 antibody [DO-1] - ChIP Grade (ab1101) at 1/1000 dilution
Lane 1 : HEK-293 (human embryonic kidney cell line) whole cell lysate
Lane 2 : DU 145 (human prostate carcinoma cell line) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution
Predicted band size: 43 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Milk before being incubated with ab1101 overnight at 4°C. Antibody binding was detected using ab175783 at a 1/10,000 dilution for 1 hour at room temperature and then imaged using the Licor Odyssey CLx.
データシートおよび資料
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参考文献 (162)
ab1101 は 162 報の論文で使用されています。
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