AKT1 Recombinant Rabbit Monoclonal Antibody [ST05-09]

RMB: 900 特惠 1500 1500

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100μl

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Catalog# ET1609-47

AKT1 Recombinant Rabbit Monoclonal Antibody [ST05-09]

Application

WB
IHC-P
IP
FC
IHC-Fr
IF-Cell

Reactivity

Human
Mouse
Rat

This product has been cited in peer reviewed publications, see list HERE

概述

产品名称

AKT1 Recombinant Rabbit Monoclonal Antibody [ST05-09]

抗体类型

Recombinant Rabbit monoclonal Antibody

免疫原

Synthetic peptide within C-terminal human AKT1.

种属反应性

Human, Mouse, Rat

验证应用

WB, IHC-P, IP, FC, IHC-Fr, IF-Cell

分子量

Predicted band size: 56 kDa

阳性对照

HeLa cell lysate, A549 cell lysate, C6 cell lysate, PC-12 cell lysate, SH-SY5Y cell lysates, mouse brain tissue, mouse kidney tissue, human kidney tissue, mouse prostate tissue, Hela, mouse hippocampus tissue, mouse cerebral cortex tissue, MCF7, C6.

偶联

unconjugated

克隆号

ST05-09

RRID

AB_3069857

产品特性

形态

Liquid

存放说明

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.

存储缓冲液

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

亚型

IgG

纯化方式

Protein A affinity purified.

应用稀释度

WB
1:5,000-1:10,000

IHC-P
1:2,000-1:5,000

FC
1:1,000

IP
1-2μg/sample

IHC-Fr
1:200

IF-Cell
1:100

发表文章中的应用

WB	查看 50 篇文献如下
IHC-P	查看 3 篇文献如下
Co-IP	查看 1 篇文献如下
IF	查看 1 篇文献如下
IP	查看 1 篇文献如下
IHC	查看 1 篇文献如下

发表文章中的种属

Human	查看 23 篇文献如下
Mouse	查看 19 篇文献如下
Rat	查看 12 篇文献如下
Goat	查看 1 篇文献如下
Rabbit	查看 1 篇文献如下
Dog	查看 1 篇文献如下

靶点

功能

The serine/threonine kinase Akt family contains several members, including Akt1 (also designated PKB or RacPK), Akt2 (also designated PKBβor RacPK-β) and Akt 3 (also designated PKB? or thyoma viral proto-oncogene 3), which exhibit sequence homology with the protein kinase A and C families and are encoded by the c-Akt proto-oncogene. All members of the Akt family have a pleckstrin homology domain. Akt1 and Akt2 are activated by PDGF stimulation. This activation is dependent on PDGFR-β tyrosine residues 740 and 751, which bind the subunit of the phosphatidylinositol 3-kinase (PI 3-kinase) complex. Activation of Akt1 by insulin or insulin-growth factor-1(IGF-1) results in phosphorylation of both Thr 308 and Ser 473. Phosphorylation of both residues is important to generate a high level of Akt1 activity, and the phosphorylation of Thr 308 is not dependent on phosphorylation of Ser 473 in vivo. Thus, Akt proteins become phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s). The activation of Akt1 and Akt2 is inhibited by the PI kinase inhibitor wortmannin, suggesting that the protein signals downstream of the PI kinases.

背景文献

1. Wang, H. et al. 2016. CSL regulates AKT to mediate androgen independence in prostate cancer progression. Prostate. 76: 140-50.

2. Wang, Z. et al. 2016. Protein 41N acts as a potential tumor suppressor linking PP1 to JNK-c-Jun pathway regulation in NSCLC. Oncotarget. 7: 509-23.

序列相似性

Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. RAC subfamily.

组织特异性

Expressed in prostate cancer and levels increase from the normal to the malignant state (at protein level). Expressed in all human cell types so far analyzed. The Tyr-176 phosphorylated form shows a significant increase in expression in breast cancers during the progressive stages i.e. normal to hyperplasia (ADH), ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC) and lymph node metastatic (LNMM) stages.

翻译后修饰

O-GlcNAcylation at Thr-305 and Thr-312 inhibits activating phosphorylation at Thr-308 via disrupting the interaction between AKT1 and PDPK1. O-GlcNAcylation at Ser-473 also probably interferes with phosphorylation at this site.; Phosphorylation on Thr-308, Ser-473 and Tyr-474 is required for full activity. Activated TNK2 phosphorylates it on Tyr-176 resulting in its binding to the anionic plasma membrane phospholipid PA. This phosphorylated form localizes to the cell membrane, where it is targeted by PDPK1 and PDPK2 for further phosphorylations on Thr-308 and Ser-473 leading to its activation. Ser-473 phosphorylation by mTORC2 favors Thr-308 phosphorylation by PDPK1. Phosphorylated at Thr-308 and Ser-473 by IKBKE and TBK1. Ser-473 phosphorylation is enhanced by interaction with AGAP2 isoform 2 (PIKE-A). Ser-473 phosphorylation is enhanced in focal cortical dysplasias with Taylor-type balloon cells. Ser-473 phosphorylation is enhanced by signaling through activated FLT3 (By similarity). Ser-473 is dephosphorylated by PHLPP. Dephosphorylated at Thr-308 and Ser-473 by PP2A phosphatase. The phosphorylated form of PPP2R5B is required for bridging AKT1 with PP2A phosphatase. Ser-473 is dephosphorylated by CPPED1, leading to termination of signaling.; Ubiquitinated via 'Lys-48'-linked polyubiquitination by ZNRF1, leading to its degradation by the proteasome (By similarity). Ubiquitinated; undergoes both 'Lys-48'- and 'Lys-63'-linked polyubiquitination. TRAF6-induced 'Lys-63'-linked AKT1 ubiquitination is critical for phosphorylation and activation. When ubiquitinated, it translocates to the plasma membrane, where it becomes phosphorylated. When fully phosphorylated and translocated into the nucleus, undergoes 'Lys-48'-polyubiquitination catalyzed by TTC3, leading to its degradation by the proteasome. Also ubiquitinated by TRIM13 leading to its proteasomal degradation. Phosphorylated, undergoes 'Lys-48'-linked polyubiquitination preferentially at Lys-284 catalyzed by MUL1, leading to its proteasomal degradation.; Acetylated on Lys-14 and Lys-20 by the histone acetyltransferases EP300 and KAT2B. Acetylation results in reduced phosphorylation and inhibition of activity. Deacetylated at Lys-14 and Lys-20 by SIRT1. SIRT1-mediated deacetylation relieves the inhibition.

亚细胞定位

Cell membrane, Cytoplasm, Membrane, Nucleus.

UNIPROT

SwissProt: P31749 Human

SwissProt: P31750 Mouse

SwissProt: P47196 Rat

别名

AKT 1 antibody

AKT antibody

AKT1 antibody

AKT1_HUMAN antibody

MGC99656 antibody

PKB antibody

PKB-ALPHA antibody

PRKBA antibody

Protein Kinase B Alpha antibody

Protein kinase B antibody

展开

图片

☑ Knockdown (KD)

Western blot analysis of AKT1 on different lysates with Rabbit anti-AKT1 antibody (ET1609-47) at 1/5,000 dilution.

Lane 1: A549-si NT cell lysate
Lane 2: A549-si AKT1 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 56 kDa
Observed band size: 56 kDa

Exposure time: 40 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1609-47) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
Western blot analysis of AKT1 on different lysates with Rabbit anti-AKT1 antibody (ET1609-47) at 1/5,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: A549 cell lysate
Lane 3: C6 cell lysate
Lane 4: PC-12 cell lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 56 kDa
Observed band size: 56 kDa

Exposure time: 43 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1609-47) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
Western blot analysis of AKT1 on SH-SY5Y cell lysates with Rabbit anti-AKT1 antibody (ET1609-47) at 1/5,000 dilution.

Lysates/proteins at 10 µg/Lane.

Predicted band size: 56 kDa
Observed band size: 56 kDa

Exposure time: 2 minutes;

10% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1609-47) at 1/5,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at /50,000 dilution was used for 1 hour at room temperature.
Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-AKT1 antibody (ET1609-47) at 1/5,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1609-47) at 1/5,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded mouse kidney tissue with Rabbit anti-AKT1 antibody (ET1609-47) at 1/5,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1609-47) at 1/5,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Application: IHC-Fr

Species: Mouse

Site: Hippocampus

Sample: Frozen section

Antibody concentration: 1/200

Antigen retrieval: The section was pre-treated using 1% SDS buffer (in PBS, pH 7.4) for 5 minutes at room temperature.
Application: IHC-Fr

Species: Mouse

Site: Cerebral cortex

Sample: Frozen section

Antibody concentration: 1/200

Antigen retrieval: The section was pre-treated using 1% SDS buffer (in PBS, pH 7.4) for 5 minutes at room temperature.
Immunocytochemistry analysis of MCF7 cells labeling AKT1 with Rabbit anti-AKT1 antibody (ET1609-47) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-AKT1 antibody (ET1609-47) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
Immunocytochemistry analysis of C6 cells labeling AKT1 with Rabbit anti-AKT1 antibody (ET1609-47) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-AKT1 antibody (ET1609-47) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
Flow cytometric analysis of MCF7 cells labeling AKT1.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1609-47, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
AKT1 was immunoprecipitated from 0.2 mg MCF7 cell lysate with ET1609-47 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1609-47 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: MCF7 cell lysate (input)
Lane 2: ET1609-47 IP in MCF7 cell lysate
Lane 3: Rabbit IgG instead of ET1609-47 in MCF7 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 23 seconds; ECL: K1801