Phospho-AKT (Ser473) Antibody

Phospho-AKT (Ser473) Monoclonal Antibody for IHC, WB, ELISA

Host / Isotype

Mouse / IgG1

Reactivity

human, mouse and More (2)

Applications

WB, IHC, IF, ELISA

Conjugate

Unconjugated

CloneNo.

1C10B8

Cat no : 66444-1-Ig

Synonyms

AKT, AKT1, AKT1-phospho-S473, pakt, p-AKT, Phospho akt, PKB, PKB ALPHA, PRKBA, Protein kinase B, Proto oncogene c Akt, RAC, RAC ALPHA, RAC PK alpha



Tested Applications

Positive WB detected inCalyculin A treated HEK-293T cells, HEK-293 cells, Jurkat cells, Calyculin A treated PC-3 cells, TPA treated Jurkat cells
Positive IHC detected inhuman breast cancer tissue
Note: suggested antigen retrieval with TE buffer pH 9.0; (*) Alternatively, antigen retrieval may be performed with citrate buffer pH 6.0

Recommended dilution

ApplicationDilution
Western Blot (WB)WB : 1:2000-1:10000
Immunohistochemistry (IHC)IHC : 1:100-1:400
Sample-dependent, check data in validation data gallery

Product Information

66444-1-Ig targets Phospho-AKT (Ser473) in WB, IHC, IF, ELISA applications and shows reactivity with human, mouse samples.

Tested Reactivity human, mouse
Cited Reactivity chicken, human, mouse, rat
Host / Isotype Mouse / IgG1
Class Monoclonal
Type Antibody
Immunogen Peptide
Full Name v-akt murine thymoma viral oncogene homolog 1
Calculated molecular weight
Observed molecular weight 60-62 kDa
GenBank accession number NM_005163
Gene symbol AKT1
Gene ID (NCBI) 207
Conjugate Unconjugated
Form Liquid
Purification Method Protein A purification
Storage Buffer PBS with 0.02% sodium azide and 50% glycerol pH 7.3.
Storage ConditionsStore at -20°C. Stable for one year after shipment. Aliquoting is unnecessary for -20oC storage.

Background Information

1) What is AKT?

The serine/threonine kinase B AKT pathway (also known as the PI3K-Akt pathway) plays a vital role in the regulation of cellular processes, including cell proliferation, survival, and growth – processes that are essential for oncogenesis. Mutation of the regulator proteins PI3K and PTEN causes uncontrolled disruption within the PI3-kinase pathway, leading to the development of human cancers (1,2; see also AKT pathway poster for more details).

2) phospho-AKT and FAQs

A)  What is the best way to normalize phosphorylated proteins analyzed by western blot?
Normalize phospho-AKT and total AKT with your loading control (e.g. Actin, tubulin), then calculate the phospho/total ratio using these normalized values.  
Put more simply:
1. Calculate the ratio of band intensities of a phospho-AKT band: the loading control.
2. Calculate the ratio of band intensities of total AKT: loading control.
3. Divide ratio obtained #1 by #2 to obtain a normalized value for comparison among different conditions. This procedure allows one to distinguish between a change in AKT expression and a change in the ratio of phospho-AKT.
* If you are looking at the differences in a phospho-AKT expression resulting from an experimental condition (e.g., knockdown), you should also show the expression of total AKT to distinguish between a change in AKT expression (transcription/translation level) and a change in the AKT phosphorylation status.
B) What is the observed molecular weight for AKT and phospho-AKT?
Molecular Weight AKT – 56 kDa
Molecular Weight phospho-AKT – 60 kDa (Figure 1)
  

Figure 1. WB: HEK-293 cell lysate was subjected to SDS PAGE followed by western blot with 60203-2-Ig (AKT antibody) and 66444-1-Ig (AKT-phospho-S473 antibody) at a dilution of 1:4000 incubated at room temperature for 1.5 hours.

C) Are there any special WB conditions to optimize staining of a phospho-AKT?
Since this is a phosphorylated protein, 5% BSA is recommended over non-fat milk as a blocking agent.
D) What are good positive and negative controls for a phospho-AKT?
- Positive Control: HEK293 cells
- Negative Control: Treatment with PI3K inhibitors (e.g. wortmannin)
E) What species does this antibody react with?

Our internal testing has confirmed that it reacts with the human and mouse forms of phospho-AKT.Reactivity with the human form is also supported by the literature’s citations of this antibody.

References:

1. Perturbations of the AKT signaling pathway in human cancer.
2. Targeting the PI3K-Akt pathway in human cancer: rationale and promise.

Protocols

Product Specific Protocols
WB protocol for Phospho-AKT (Ser473) antibody 66444-1-IgDownload protocol
IHC protocol for Phospho-AKT (Ser473) antibody 66444-1-IgDownload protocol
Standard Protocols
Click here to view our Standard Protocols

Publications

SpeciesApplicationTitle
humanWB

Med Sci Monit

Everolimus Reverses Palbociclib Resistance in ER+ Human Breast Cancer Cells by Inhibiting Phosphatidylinositol 3-Kinase(PI3K)/Akt/Mammalian Target of Rapamycin (mTOR) Pathway.

Authors - Lin Chen
ratWB,IF

Neural Regen Res

Neuroprotective effects of rapamycin on spinal cord injury in rats by increasing autophagy and Akt signaling.

Authors - Xi-Gong Li
humanWB

Cancer Cell Int

Overexpression of CD59 inhibits apoptosis of T-acute lymphoblastic leukemia via AKT/Notch1 signaling pathway.

Authors - Yanfei Jia
humanWB

Biochem Biophys Res Commun

Long non-coding RNA FAM84B-AS promotes resistance of gastric cancer to platinum drugs through inhibition of FAM84B expression.

Authors - Yeqian Zhang
humanWB

Br J Pharmacol

Potent effects of dioscin against hepatocellular carcinoma through regulating TIGAR-mediated apoptosis, autophagy and DNA damage.

Authors - Zhang Mao
mouseWB

Front Physiol

Autophagy Protects the Blood-Brain Barrier Through Regulating the Dynamic of Claudin-5 in Short-Term Starvation.

Authors - Zhenguo Yang