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Phospho-AKT (Ser473) Monoclonal antibody, PBS Only (Capture)

Phospho-AKT (Ser473) Monoclonal Antibody for WB, IHC, FC (Intra), Cytometric bead array, Indirect ELISA

Host / Isotype

Mouse / IgG1

Reactivity

human, mouse, rat

Applications

WB, IHC, FC (Intra), Cytometric bead array, Indirect ELISA

Conjugate

Unconjugated

CloneNo.

1C10B8

Cat no : 66444-1-PBS

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Synonyms

AKT pan, AKT 1, AKT (pan), AKT, AKT1



Planning an IHC experiment? We recommend our IHCeasy AKT Ready-To-Use IHC Kit. AKT primary antibody included.

产品信息

66444-1-PBS targets Phospho-AKT (Ser473) as part of a matched antibody pair:

MP50177-1: 66444-1-PBS capture and 60203-2-PBS detection (validated in N/A)

Unconjugated mouse monoclonal antibody pair in PBS only (BSA and azide free) storage buffer at a concentration of 1 mg/mL, ready for conjugation.

This conjugation ready format makes antibodies ideal for use in many applications including: ELISAs, multiplex assays requiring matched pairs, mass cytometry, and multiplex imaging applications.Antibody use should be optimized by the end user for each application and assay.

Tested Applications WB, IHC, FC (Intra), Cytometric bead array, Indirect ELISA Application Description
Tested Reactivity human, mouse, rat
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 pan, AKT 1, AKT (pan), AKT, AKT1
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
UNIPROT IDP31749
Storage Buffer PBS Only
Storage ConditionsStore at -80°C.
The product is shipped with ice packs. Upon receipt, store it immediately at -80°C

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.