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Phospho-AKT (Ser473) Monoclonal antibody
Phospho-AKT (Ser473) Monoclonal Antibody for WB, IHC, FC (Intra), ELISA
Host / Isotype
Mouse / IgG1
Reactivity
human, mouse, rat and More (6)
Applications
WB, IHC, FC (Intra), ELISA and More (2)
Conjugate
Unconjugated
1039
CloneNo.
1C10B8
验证数据展示
经过测试的应用
Positive WB detected in | Calyculin A treated PC-3 cells, Calyculin A treated HEK-293T cells, TPA treated Jurkat cells, HSC-T6 cells, Calyculin A treated HSC-T6 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 (Intra) detected in | Calyculin A treated PC-3 cells |
推荐稀释比
Application | Dilution |
---|---|
Western Blot (WB) | WB : 1:2000-1:10000 |
Immunohistochemistry (IHC) | IHC : 1:100-1:400 |
Flow Cytometry (FC) (INTRA) | FC (INTRA) : 0.50 ug per 10^6 cells in a 100 µl suspension |
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. |
产品信息
66444-1-Ig targets Phospho-AKT (Ser473) in WB, IHC, IF, FC (Intra), IP, ELISA applications and shows reactivity with human, mouse, rat samples.
Tested Applications | WB, IHC, FC (Intra), ELISA Application Description |
Cited Applications | WB, IHC, IF, IP |
Tested Reactivity | human, mouse, rat |
Cited Reactivity | human, mouse, rat, pig, rabbit, canine, chicken, zebrafish, duck |
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 (Ser473), P AKT, P AKT (Ser473), P-AKT, P-AKT (Ser473) |
Observed Molecular Weight | 60-62 kDa |
GenBank Accession Number | NM_005163 |
Gene Symbol | AKT1 |
Gene ID (NCBI) | 207 |
RRID | AB_2782958 |
Conjugate | Unconjugated |
Form | Liquid |
Purification Method | Protein A purification |
UNIPROT ID | P31749 |
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. |
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.
实验方案
Product Specific Protocols | |
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WB protocol for Phospho-AKT (Ser473) antibody 66444-1-Ig | Download protocol |
IHC protocol for Phospho-AKT (Ser473) antibody 66444-1-Ig | Download protocol |
Standard Protocols | |
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Click here to view our Standard Protocols |
发表文章
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