Anticorps Monoclonal anti-Phospho-AKT (Ser473)

Phospho-AKT (Ser473) Monoclonal Antibody for FC (Intra)

Hôte / Isotype

Mouse / IgG1

Réactivité testée

Humain, souris

Applications

FC (Intra)

Conjugaison

CoraLite®594 Fluorescent Dye

CloneNo.

1C10B8

N° de cat : CL594-66444

Synonymes

AKT, AKT1, Phospho-AKT (Ser473), PKB, PKB ALPHA, PRKBA, Protein kinase B, Proto oncogene c Akt, RAC, RAC ALPHA, RAC PK alpha

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Galerie de données de validation

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  FC experiment of PC-3 using CL594-66444

  1X10^6 PC-3 cells untreated (dashed line) or treated with Calyculin A (red) were intracellularly stained with 0.25 ug CoraLite®594 Anti-Human Phospho-AKT (Ser473) (CL594-66444, Clone:1C10B8), or 0.25 ug Control Antibody (blue). Cells were fixed and permeabilized with True-Nuclear Transcription Factor Buffer Set.

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Applications testées

• Résultats positifs en cytométrie: cellules PC-3 traitées à la calyculine A

Dilution recommandée

• Flow Cytometry (FC): FC : 0.25 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

Informations sur le produit

CL594-66444 cible Phospho-AKT (Ser473) dans les applications de FC (Intra) et montre une réactivité avec des échantillons Humain, souris

• Réactivité: Humain, souris
• Hôte / Isotype: Mouse / IgG1
• Clonalité: Monoclonal
• Type: Anticorps
• Immunogène: Peptide
• Nom complet: v-akt murine thymoma viral oncogene homolog 1
• Poids moléculaire observé: 60-62 kDa
• Numéro d’acquisition GenBank: NM_005163
• Symbole du gène: AKT1
• Identification du gène (NCBI): 207
• Conjugaison: CoraLite®594 Fluorescent Dye
• Excitation/Emission maxima wavelengths: 588 nm / 604 nm
• Forme: Liquide
• Méthode de purification: Purification par protéine A
• Tampon de stockage: PBS avec glycérol à 50 %, Proclin300 à 0,05 % et BSA à 0,5 %, pH 7,3.
• Conditions de stockage: Stocker à -20 °C. Éviter toute exposition à la lumière. L'aliquotage n'est pas nécessaire pour le stockage à -20^oC Les 20ul contiennent 0,1% de BSA.

Informations générales

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.