• Featured Product
  • KD/KO Validated

Phospho-AKT (Ser473) Monoclonal antibody

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

Host / Isotype

Mouse / IgG1


human, mouse, rat and More (5)







Cat no : 66444-1-Ig


AKT, AKT1, pakt, 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 PC-3 cells, Jurkat cells, HSC-T6 cells, NIH/3T3 cells, Calyculin A treated HEK-293T cells, TPA treated Jurkat cells, Calyculin A treated Jurkat cells, Calyculin A treated HEK-293 cells, Calyculin A treated HSC-T6 cells, Calyculin A treated NIH/3T3 cells
Positive IHC detected inhuman breast cancer tissue, Calyculin A treated Jurkat cells, human colon cancer tissue
Note: suggested antigen retrieval with TE buffer pH 9.0; (*) Alternatively, antigen retrieval may be performed with citrate buffer pH 6.0
Positive FC detected inCalyculin A treated PC-3 cells

Recommended dilution

Western Blot (WB)WB : 1:2000-1:10000
Immunohistochemistry (IHC)IHC : 1:100-1:400
Flow Cytometry (FC)FC : 0.50 ug per 10^6 cells in a 100 µl suspension
It is recommended that this reagent should be titrated in each testing system to obtain optimal results.
Sample-dependent, Check data in validation data gallery.

Product Information

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

Tested Reactivity human, mouse, rat
Cited Reactivityhuman, mouse, rat, chicken, rabbit, zebrafish, pig, canine
Host / Isotype Mouse / IgG1
Class Monoclonal
Type Antibody
Immunogen Peptide
Full Name v-akt murine thymoma viral oncogene homolog 1
Observed Molecular Weight 60-62 kDa
GenBank Accession NumberNM_005163
Gene Symbol AKT1
Gene ID (NCBI) 207
Conjugate Unconjugated
Form Liquid
Purification MethodProtein 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. 20ul sizes contain 0.1% BSA.

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.


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


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



Cell Res

Inhibiting Hv1 channel in peripheral sensory neurons attenuates chronic inflammatory pain and opioid side effects.

Authors - Qiansen Zhang

Cell Res

In vivo self-assembled small RNAs as a new generation of RNAi therapeutics.

Authors - Zheng Fu

Nat Commun

Genome-wide enhancer-gene regulatory maps link causal variants to target genes underlying human cancer risk

Authors - Pingting Ying

Sci Adv

Platelet P-selectin initiates cross-presentation and dendritic cell differentiation in blood monocytes.

Authors - Patrick Han

Acta Pharm Sin B

Celastrol targets adenylyl cyclase-associated protein 1 to reduce macrophages-mediated inflammation and ameliorates high fat diet-induced metabolic syndrome in mice.

Authors - Yuyu Zhu

J Pineal Res

Melatonin and verteporfin synergistically suppress the growth and stemness of head and neck squamous cell carcinoma through the regulation of mitochondrial dynamics

Authors - Ye Young Shin


The reviews below have been submitted by verified Proteintech customers who received an incentive forproviding their feedback.


András (Verified Customer) (07-31-2023)

The Mk-2206 is an allosteric Akt inhibitor that prevents its recruitment to the membrane and consequently its phosphorylation. The Capivasertib is an Akt competitive inhibitor which induces its over phosphorylation.

  • Applications: Western Blot
  • Cell Tissue Type: P19 derived neurons.
Phospho-AKT (Ser473) Antibody Western Blot validation ( dilution) in P19 derived neurons. (Cat no:66444-1-Ig)

Jorge (Verified Customer) (07-26-2022)

Good signal. Unspecific band below 100 kDa. Used PageRuler Plus Prestained Protein Ladder and chemiluminescence was detected in the 70 kDa marker.

  • Applications: Western Blot
  • Primary Antibody Dilution: 1:2000
  • Cell Tissue Type: White adipose tissue

Tom (Verified Customer) (12-15-2020)

10ug total protein of HEK293T lysate loaded. Membrane blocked in 5% BSA. Antibody (1:1,000) incubated overnight in block at 4 degrees. Anti-mouse HRP used at 1 in 10,000 to detect band.

  • Applications: Western Blot
  • Primary Antibody Dilution: 1:1000
  • Cell Tissue Type: HEK293T
Phospho-AKT (Ser473) Antibody Western Blot validation (1:1000 dilution) in HEK293T (Cat no:66444-1-Ig)