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Phospho-AKT (Ser473) Monoclonal antibody

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

Host / Isotype

Mouse / IgG1

Reactivity

human, mouse, rat and More (6)

Applications

WB, IHC, FC (Intra), ELISA and More (2)

Conjugate

Unconjugated

CloneNo.

1C10B8

Cat no : 66444-1-Ig

Print datasheet

Synonyms

AKT (Ser473), P AKT, P AKT (Ser473), P-AKT, P-AKT (Ser473)



经过测试的应用

Positive WB detected inCalyculin A treated PC-3 cells, Calyculin A treated HEK-293T cells, TPA treated Jurkat cells, HSC-T6 cells, Calyculin A treated HSC-T6 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 (Intra) detected inCalyculin A treated PC-3 cells
Planning an IHC experiment? We recommend our IHCeasy AKT Ready-To-Use IHC Kit. AKT primary antibody included.

推荐稀释比

ApplicationDilution
Western Blot (WB)WB : 1:2000-1:10000
Immunohistochemistry (IHC)IHC : 1:100-1:400
Flow Cytometry (FC) (INTRA)FC (INTRA) : 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.

产品信息

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

Tested Applications WB, IHC, FC (Intra), ELISA Application Description
Cited ApplicationsWB, IHC, IF, IP
Tested Reactivity human, mouse, rat
Cited Reactivityhuman, mouse, rat, pig, rabbit, canine, chicken, zebrafish, duck
Immunogen Peptide 种属同源性预测
Host / Isotype Mouse / IgG1
Class Monoclonal
Type Antibody
Full Name v-akt murine thymoma viral oncogene homolog 1
Synonyms AKT (Ser473), P AKT, P AKT (Ser473), P-AKT, P-AKT (Ser473)
Observed Molecular Weight 60-62 kDa
GenBank Accession NumberNM_005163
Gene Symbol AKT1
Gene ID (NCBI) 207
RRIDAB_2782958
Conjugate Unconjugated
Form Liquid
Purification MethodProtein A purification
UNIPROT IDP31749
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.

背景介绍

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.

实验方案

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

发表文章

SpeciesApplicationTitle

Adv Mater

Targeted Macrophage CRISPR-Cas13 Mrna Editing in Immunotherapy for Tendon Injury

Authors - Shuo Wang
mouseWB,IHC

Cell Res

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

Authors - Zheng Fu
mouseIF

Cell Res

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

Authors - Qiansen Zhang
human,mouseWB

Nat Commun

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

Authors - Pingting Ying
humanWB

Sci Adv

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

Authors - Patrick Han
mouseWB

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