AKT1/2/3 Recombinant Rabbit Monoclonal Antibody [ST48-09] - BSA and Azide free
Catalog# HA750179
AKT1/2/3 Recombinant Rabbit Monoclonal Antibody [ST48-09] - BSA and Azide free
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WB
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IF-Cell
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IF-Tissue
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IHC-P
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IP
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FC
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IHC-Fr
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Human
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Mouse
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Rat
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Monkey
Overview
Product Name
AKT1/2/3 Recombinant Rabbit Monoclonal Antibody [ST48-09] - BSA and Azide free
Antibody Type
Recombinant Rabbit monoclonal Antibody
Immunogen
Recombinant protein within human AKT3 aa 300-479.
Species Reactivity
Human, Mouse, Rat, Monkey
Validated Applications
WB, IF-Cell, IF-Tissue, IHC-P, IP, FC, IHC-Fr
Molecular Weight
Predicted band size: 56 kDa
Positive Control
MCF7 cell lysate, U-2 OS cell lysate, Jurkat cell lysate, C6 cell lysate, mouse heart tissue lysate, mouse testis tissue lysate, rat heart tissue lysate, rat testis tissue lysate, MCF7, RAW264.7, C6, human brain tissue, human lung tissue, mouse brain tissue, mouse lung tissue, rat brain tissue, rat lung tissue, mouse hippocampus tissue, mouse cerebral cortex tissue.
Conjugation
unconjugated
Clone Number
ST48-09
Product Features
Form
Liquid
Concentration
1ug/ul
Storage Instructions
Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage Buffer
PBS (pH7.4).
Isotype
IgG
Purification Method
Protein A affinity purified.
Application Dilution
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WB
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1:5,000-1:10,000
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IF-Cell
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1:100-1:200
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IF-Tissue
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1:500-1:1,000
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IHC-P
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1:2,000
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FC
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1:1,000
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IP
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1-2μg/sample
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IHC-Fr
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1:100
Target
Function
The serine/threonine kinase Akt family contains several members, including Akt1 (also designated PKB or RacPK), Akt2 (also designated PKBβ or RacPK-β) and Akt 3 (also designated PKBγ 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.
Background References
1. Buendia, I. et al. 2015. The Melatonin-N,N-Dibenzyl(N-methyl)amine Hybrid ITH91/IQM157 Affords Neuroprotection in an in Vitro Alzheimer\'s Model via Hemo-oxygenase-1 Induction. ACS chemical neuroscience. 6: 288-96.
2. Siendones, E. et al. 2014. Membrane-bound CYB5R3 is a common effector of nutritional and oxidative stress response through FOXO3a and Nrf2. Antioxidants & redox signaling. 21: 1708-25.
Sequence Similarity
Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. RAC subfamily.
Tissue Specificity
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.
Post-translational Modification
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.
Subcellular Location
Cell membrane, Cytoplasm, Membrane, Nucleus.
UNIPROT
Synonyms
AKT antibody
AKT1 antibody
AKT1 kinase antibody
AKT1m antibody
AKT2 antibody
AKT2 kinase antibody
Akt3 antibody
AKT3_HUMAN antibody
CAKT antibody
CWS6 antibody
ExpandImages
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Western blot analysis of AKT1/2/3 on different lysates with Rabbit anti-AKT1/2/3 antibody (HA750179) at 1/5,000 dilution.
Lane 1: MCF7 cell lysate (20 µg/Lane)
Lane 2: A549 cell lysate (20 µg/Lane)
Lane 3: U-2 OS cell lysate (20 µg/Lane)
Lane 4: COS-1 cell lysate (20 µg/Lane)
Lane 5: NIH/3T3 cell lysate (20 µg/Lane)
Lane 6: RAW264.7 cell lysate (20 µg/Lane)
Lane 7: C6 cell lysate (20 µg/Lane)
Lane 8: PC-12 cell lysate (20 µg/Lane)
Lane 9: Mouse brain tissue lysate (20 µg/Lane)
Lane 10: Mouse heart tissue lysate (20 µg/Lane)
Lane 11: Mouse testis tissue lysate (20 µg/Lane)
Lane 12: Rat brain tissue lysate (20 µg/Lane)
Lane 13: Rat heart tissue lysate (20 µg/Lane)
Lane 14: Rat testis tissue lysate (20 µg/Lane)
Predicted band size: 56 kDa
Observed band size: 56 kDa
Exposure time: 24 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 (HA750179) 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 1/50,000 dilution was used for 1 hour at room temperature. -
AKT1/2/3 was immunoprecipitated from 0.2 mg MCF7 cell lysate with HA750179 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using HA750179 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: HA750179 IP in MCF7 cell lysate
Lane 3: Rabbit IgG instead of HA750179 in MCF7 cell lysate
Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 23 seconds; ECL: K1801 -
Immunocytochemistry analysis of MCF7 cells labeling AKT1/2/3 with Rabbit anti-AKT1/2/3 antibody (HA750179) 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-AKT1/2/3 antibody (HA750179) 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 RAW264.7 cells labeling AKT1/2/3 with Rabbit anti-AKT1/2/3 antibody (HA750179) 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-AKT1/2/3 antibody (HA750179) 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 AKT1/2/3 with Rabbit anti-AKT1/2/3 antibody (HA750179) 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-AKT1/2/3 antibody (HA750179) 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. -
Immunohistochemical analysis of paraffin-embedded human brain tissue with Rabbit anti-AKT1/2/3 antibody (HA750179) at 1/2,000 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 (HA750179) at 1/2,000 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 human lung tissue with Rabbit anti-AKT1/2/3 antibody (HA750179) at 1/2,000 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 (HA750179) at 1/2,000 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 brain tissue with Rabbit anti-AKT1/2/3 antibody (HA750179) at 1/2,000 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 (HA750179) at 1/2,000 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 lung tissue with Rabbit anti-AKT1/2/3 antibody (HA750179) at 1/2,000 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 (HA750179) at 1/2,000 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 rat brain tissue with Rabbit anti-AKT1/2/3 antibody (HA750179) at 1/2,000 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 (HA750179) at 1/2,000 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 rat lung tissue with Rabbit anti-AKT1/2/3 antibody (HA750179) at 1/2,000 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 (HA750179) at 1/2,000 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: 1/100
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: 1/100
Antigen retrieval: The section was pre-treated using 1% SDS buffer (in PBS, pH 7.4) for 5 minutes at room temperature. -
Flow cytometric analysis of MCF7 cells labeling AKT1/2/3.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA750179, 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). -
Flow cytometric analysis of RAW264.7 cells labeling AKT1/2/3.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA750179, 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). -
Flow cytometric analysis of C6 cells labeling AKT1/2/3.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA750179, 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).
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
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