HRP-conjugated Phospho-AKT (Ser473) Monoclonal antibody

Phospho-AKT (Ser473) Monoclonal Antibody for WB

Host / Isotype

Mouse / IgG1

Reactivity

human, mouse

Applications

WB

Conjugate

HRP

CloneNo.

1C10B8

Cat no : HRP-66444

Synonyms

AKT, AKT1, PKB, PKB ALPHA, PRKBA, Protein kinase B, Proto oncogene c Akt, RAC, RAC ALPHA, RAC PK alpha

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Validation Data Gallery

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  WB analysis of HEK-293 using HRP-66444

  Untreated and Calyculin A treated HEK-293 cells were subjected to SDS PAGE followed by western blot with HRP-66444 (Phospho-AKT (Ser473) antibody) at dilution of 1:16000 incubated at room temperature for 1.5 hours.

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Tested Applications

• Positive WB detected in: Calyculin A treated HEK-293 cells

Recommended dilution

• Western Blot (WB): WB : 1:5000-1:50000

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

HRP-66444 targets Phospho-AKT (Ser473) in WB applications and shows reactivity with human, mouse samples.

• Tested Reactivity: human, mouse
• 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 Number: NM_005163
• Gene Symbol: AKT1
• Gene ID (NCBI): 207
• Conjugate: HRP
• Form: Liquid
• Purification Method: Protein A purification
• Storage Buffer: PBS with 50% Glycerol, 0.05% Proclin300, 0.5% BSA, pH 7.3.
• Storage Conditions: Store at -20°C. Avoid exposure to light. Stable for one year after shipment. Aliquoting is unnecessary for -20^oC 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.