p53 抗体 (Tumor Protein P53) (meLys370)

Details for Product anti-TP53 Antibody No. ABIN4902067, 供应商: Log in to see
抗原
  • tp53
  • p53
  • TP53
  • Tp53
  • bbl
  • bfy
  • bhy
  • p44
  • TpMs
  • BCC7
  • LFS1
  • P53
  • TRP53
  • Trp53
  • Xp53
  • brp53
  • drp53
  • etID22686.5
  • fb40d06
  • wu:fb40d06
  • zgc:111919
  • cellular tumor antigen p53
  • tumor protein p53
  • tumor supressor p53
  • p53 tumor suppressor
  • transformation related protein 53
  • transformation related protein 53, pseudogene
  • trichoplein, keratin filament binding
  • Wistar clone pR53P1 p53 pseudogene
  • CpipJ_CPIJ002758
  • TP53
  • tp53
  • P53
  • Trp53
  • Trp53-ps
  • TCHP
  • Tp53
  • p53-ps
抗原表位
meLys370
173
128
94
84
74
71
70
62
59
55
50
49
48
47
42
38
32
31
29
28
24
22
20
20
17
16
15
15
15
15
15
14
12
11
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10
9
9
8
7
7
7
7
7
7
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6
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5
5
5
5
5
5
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
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1
1
1
1
1
1
1
1
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1
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1
1
1
1
1
1
1
1
1
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1
1
1
1
适用

2428
519
426
188
127
94
87
84
35
23
20
18
17
16
15
14
6
4
4
4
4
3
3
3
2
2
1
1
宿主

1371
1170
7
3
1
1
1
1
克隆类型
多克隆
标记
This p53 antibody is un-conjugated
81
78
65
55
49
48
34
34
26
26
25
25
25
25
25
24
24
24
23
22
22
21
14
14
14
14
14
12
7
5
5
5
5
5
5
5
5
4
3
2
2
2
应用范围
ELISA, Western Blotting (WB)
1931
771
751
742
651
637
435
347
316
179
72
41
32
31
24
17
17
15
13
9
9
4
4
3
3
3
2
2
1
1
1
1
1
1
1
1
1
1
选项
Supplier
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Supplier Product No.
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'独立验证'标志
抗原 p53K370me
Lot Number B1001
Method validated Western Blotting
Positive Control

p53, in vitro specifically p53K370me1 methylated using methyltransferase SMYD2

ectopic SMYD-methylated wildtype p53 from 293T cell extracts

Negative Control

p53, in vitro methylated using methyltransferases G9a and SETD8

p53ΔCTD, lacking C-terminal domain containing known methylable lysines

p53K370R, lacking the lysine 370 methylation site, mutated into arginine

Notes

Passed. ABIN4902067 specifically recognizes monomethylated p53K370me1 and to a lesser extent p53Kme1.

Primary Antibody ABIN4902067
Secondary Antibody HRP-conjugated goat anti-rabbit or anti-mouse antibody (Biorad)
实验流程
  • In vitro p53 methylation assay:
    • Clone SMYD2 and p53 from human cDNA library into pGex6p1 vector.
    • Perform p53 mutagenesis using QuickChange II site-directed mutagenesis kit (Agilent, 200523).
    • Transform vectors into BL21 pLys bacteria (Agilent) and induced protein expression with 0.1mM IPTG in 250mL culture ON at 19°C.
    • Purify proteins by GST pulldown and quantify using Bradford assay and Coomassie gel staining.
    • For the in vitro methylation assays incubate 1 to 2µg recombinant protein with 1µg of recombinant methyltransferases and 2µCi 3H-AdoMet (American Radiolabelled Chemicals) in buffer containing 50mM Tris-HCl pH8.0, 10% glycerol, 20mM KCl, 5mM MgCl2, and 1mMPMSF at ON at 30°C.
    • Resolve the reaction mixture by SDS–PAGE, followed by autoradiography and Coomassie stain (Pierce).
  • "In cellulo” p53 methylation assay:
    • Subclone SMYD2 and p53 constructs from pGex6p1 vectors into pcagFLAG (SMYD2) or pCDNA3.1HA (p53).
    • Transfect plasmids into 293T cells using Lipofectamine 2000 (ThermoFisher) according to supplier’s protocol.
    • Harvest cells 36h post-transfection to allow methylation of ectopic p53 by SMYD2 in cells.
  • Western blot:
    • Separate 20µg of each sample on freshly cast SDS-PAGE gel in a gel electrophoresis chamber (BioRad) for 1h at 140V and RT.
    • Transfer proteins onto ethanol-activated 0.2µm Hybond PVDF membrane (Amersham) with a semi-dry Trans-Blot Turbo (BioRad) for 30min at 20V.
    • Block the membrane using 5% milk in TBS-Tween 0.1% for 1h at RT.
    • Incubate the membrane with primary rabbit anti-p53 meLys370 antibody (antibodies-online, ABIN4902067, LOT B1001), mouse anti-HA antibody (Sigma), or mouse anti-Flag tag antibody (Sigma) diluted 1:1000 in 1% BSA TBS-Tween 0.1% ON at 4°C.
    • Wash the membrane 3x for 10min in TBS-Tween 0.1%.
    • Incubate the membrane with secondary HRP-conjugated goat anti-rabbit or anti-mouse antibody (Biorad) diluted 1:3000 in 1% BSA TBS-Tween 0.1% for 1h at RT.
    • Wash the membrane 3x for 10min in TBS-Tween 0.1%.
    • Visualize protein bands using ECL RevelBlot intense (Ozyme) and signal detection on Hyperfilm ECL 18 x24cm (Amersham).
Experimental Notes
  • Antibody ABIN4902067 recognizes greatly its specific substrate in vitro: it shows a strong signal when SMYD2 is present, implying monomethylation of K370. The signal is lost when using p53K370R and p53ΔCTD mutants. However, ABIN4902067 also recognizes to a lesser extent in vitro other p53 methylation such as p53 K373me1/2 and p53K382me2 caused by G9a and SETD8 respectively.
  • The antibody does recognize unmethylated p53 when using a large quantity of the recombinant protein (not shown).
  • ABIN4902067 gives a very clean and specific signal when using ectopic p53 from 293T cell extract: only p53 methylated by SMYD2 is detected, not the p53K370R mutant. The antibody can even be used without an initial immunoprecipitation of total p53 as observable in our figure.
  • The binding affinity of ABIN4902067 can be ordered as follows: p53K370me1>p53Kme1>>>p53.
Validation Images
Western Blotting validation image for anti-Tumor Protein P53 (TP53) (meLys370) antibody (ABIN4902067)

A. In vitro methylation assay of wt p53 and the p53K370R and p53ΔC...

亚型 IgG
特异性 Mono-Methyl-p53 (K370) Polyclonal Antibody detects endogenous levels of p53 around the methylation site of K370 protein.
产品特性 Rabbit Polyclonal to p53 (Mono Methyl Lys370).
纯化方法 The antibody was affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogen.
别名 p53 (TP53 Antibody 摘要)
基因ID 7157
UniProt P04637
研究领域 Transcription Factors, Apoptosis/Necrosis, DNA/RNA, Cell Cycle, Cancer
途径 p53 Pathway, MAPK Pathway, PI3K-Akt Signaling, Apoptosis, AMPK Signaling, Chromatin Binding, ER-Nucleus Signaling, Positive Regulation of Endopeptidase Activity, Hepatitis C, Protein targeting to Nucleus, Autophagy
应用备注 WB 1:500-1:2000, ELISA 1:10000
限制 仅限研究用
状态 Liquid
浓度 1 mg/mL
缓冲液 Liquid in PBS containing 50 % glycerol, 0.5 % BSA and 0.02 % sodium azide.
储存液 Sodium azide
注意事项 This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
注意事项 Avoid repeated freeze/thaw cycles.
储存条件 -20 °C
储存方法 Store at -20°C.
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