Phospho-AKT (Ser473) Monoclonal antibody
Phospho-AKT (Ser473) Monoclonal Antibody for FC, IHC, WB, ELISA
Host / Isotype
Mouse / IgG1
human, mouse, rat and More (4)
WB, IHC, IF, FC, 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|
|KD/KO||See 1 publications below|
|WB||See 695 publications below|
|IHC||See 52 publications below|
|IF||See 9 publications below|
|FC||See 1 publications below|
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, canine, chicken, pig, 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. 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.
1. Perturbations of the AKT signaling pathway in human cancer.
2. Targeting the PI3K-Akt pathway in human cancer: rationale and promise.
|Product Specific Protocols|
|WB protocol for Phospho-AKT (Ser473) antibody 66444-1-Ig||Download protocol|
|IHC protocol for Phospho-AKT (Ser473) antibody 66444-1-Ig||Download protocol|
|Click here to view our Standard Protocols|
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The reviews below have been submitted by verified Proteintech customers who received an incentive forproviding their feedback.
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.