Phospho-AKT (Ser473) Monoclonal antibody
Phospho-AKT (Ser473) Monoclonal Antibody for FC, IHC, WB, ELISA
Cat no : 66444-1-Ig
Validation Data Gallery
|Positive WB detected in||Calyculin A treated Jurkat cells, Calyculin A treated HEK-293T cells, Calyculin A treated PC-3 cells, Calyculin A treated HEK-293 cells, TPA treated Jurkat cells, Calyculin A treated HSC-T6 cells, Calyculin A treated NIH/3T3 cells|
|Positive IHC detected in||human 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 detected in||Calyculin A treated PC-3 cells|
|Western Blot (WB)||WB : 1:5000-1:50000|
|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, FC, ELISA applications and shows reactivity with human, mouse, rat samples.
|Tested Reactivity||human, mouse, rat|
|Cited Reactivity||human, mouse, rat, chicken, rabbit, zebrafish, pig, canine|
|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. 20ul sizes contain 0.1% BSA.|
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.
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The reviews below have been submitted by verified Proteintech customers who received an incentive forproviding their feedback.
András (Verified Customer) (07-31-2023)
The Mk-2206 is an allosteric Akt inhibitor that prevents its recruitment to the membrane and consequently its phosphorylation. The Capivasertib is an Akt competitive inhibitor which induces its over phosphorylation.
Jorge (Verified Customer) (07-26-2022)
Good signal. Unspecific band below 100 kDa. Used PageRuler Plus Prestained Protein Ladder and chemiluminescence was detected in the 70 kDa marker.
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.