|Positive WB detected in||Calyculin A treated HEK-293T cells, HEK-293 cells, Jurkat cells, Calyculin A treated PC-3 cells, TPA treated Jurkat cells|
|Positive IHC detected in||human breast cancer tissue|
Note: suggested antigen retrieval with TE buffer pH 9.0; (*) Alternatively, antigen retrieval may be performed with citrate buffer pH 6.0
|Western Blot (WB)||WB : 1:2000-1:10000|
|Immunohistochemistry (IHC)||IHC : 1:100-1:400|
|Sample-dependent, check data in validation data gallery|
66444-1-Ig targets Phospho-AKT (Ser473) in WB, IHC, IF,ELISA applications and shows reactivity with human, mouse samples.
|Tested Reactivity||human, mouse|
|Cited Reactivity||canine, chicken, human, mouse, rat, swine, zebrafish|
|Host / Isotype||Mouse / IgG1|
|Full Name||v-akt murine thymoma viral oncogene homolog 1|
|Observed molecular weight||60-62 kDa|
|GenBank accession number||NM_005163|
|Gene ID (NCBI)||207|
|Purification Method||Protein A purification|
|Storage Buffer||PBS with 0.02% sodium azide and 50% glycerol pH 7.3.|
|Storage Conditions||Store at -20°C. Stable for one year after shipment. Aliquoting is unnecessary for -20oC storage.|
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.
Med Sci Monit
Neural Regen Res
Neuroprotective effects of rapamycin on spinal cord injury in rats by increasing autophagy and Akt signaling.
Cancer Cell Int
Overexpression of CD59 inhibits apoptosis of T-acute lymphoblastic leukemia via AKT/Notch1 signaling pathway.
Biochem Biophys Res Commun
Long non-coding RNA FAM84B-AS promotes resistance of gastric cancer to platinum drugs through inhibition of FAM84B expression.
Br J Pharmacol
Potent effects of dioscin against hepatocellular carcinoma through regulating TIGAR-mediated apoptosis, autophagy and DNA damage.
Autophagy Protects the Blood-Brain Barrier Through Regulating the Dynamic of Claudin-5 in Short-Term Starvation.
The reviews below have been submitted by verified Proteintech customers who received an incentive forproviding their feedback.
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.