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Anti-Phospho-Akt (Ser473) Rabbit Antibody [B19D19]

Catalog No.: F3507

Application: Reactivity: Human, Mouse, Rat

Usage Information

Dilution
WB1:1000 IP1:200 IF1:100 - 1:200 FCM1:200 - 1:800

Application
WB, IP, IF, FCM

Reactivity
Human, Mouse, Rat

Source
Rabbit

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
60 kDa

Positive Control	SK-MEL-28 (human Insulin, 100 nM, 20 min); NIH/3T3 (mouse PDGF-BB, 100 ng/mL, 20 min); H-4-II-E (human IGF-1, 100 ng/mL, 5 min); NIH/3T3 (PDGF-treated and pretreated with wortmannin/rapamycin); 3T3 (PDGF, 100ng/mL, 15 min); MCF-7 (hIGF-1, 100ng/ml, 10 mins)
Negative Control	SK-MEL-28; NIH/3T3; H-4-II-E; 3T3; MCF-7

Datasheet & SDS

Biological Description

Specificity
Phospho-Akt (Ser473) Rabbit mAb detects endogenous levels of Akt1 only when phosphorylated at Ser473. This antibody also recognizes Akt2 and Akt3 when phosphorylated at the corresponding residues.

Clone
B19D19

Synonym(s)
RAC-beta serine/threonine-protein kinase, Protein kinase Akt-2, Protein kinase B beta (PKB beta), RAC protein kinase beta, AKT2, RAC-gamma serine/threonine-protein kinase, Protein kinase Akt-3, Protein kinase B gamma (PKB gamma), RAC-PK-gamma, STK-2, AKT3, PKBG, RAC-alpha serine/threonine-protein kinase, Protein kinase B (PKB), Protein kinase B alp

Background
Akt—also known as protein kinase B (PKB) or Rac—is a pivotal regulator of cell survival and apoptosis. It is activated by insulin as well as various growth and survival factors via a PI3 kinase–dependent pathway that is sensitive to wortmannin. Of its three isoforms, Akt1, Akt2, and Akt3, Akt1 has been studied most extensively in the contexts of cancer and metabolism. Akt1 is a central effector in the phosphatidylinositol 3,4,5-triphosphate (PIP3) signaling cascade and belongs to the AGC family of over 60 protein kinases, many of which are regulated through phosphorylation at both the activation loop and the C-terminal tail. In some AGC kinases, the C-terminal phosphorylation site is naturally replaced by an acidic aspartate, and for Akt1, the S473D substitution mimics the phosphorylated Ser473 state. In addition to the canonical Ser473 phosphorylation, Akt1 can also be phosphorylated at Ser477 and Thr479 by Cdk2/cyclinA2 within the nucleus. These non-canonical C-terminal modifications have been proposed to activate Akt1 in the nuclear compartment and promote cell-cycle progression. Mechanistic studies using Asp/Glu substitutions and peptide complementation suggest these modifications activate Akt1 through a process similar to Ser473 phosphorylation. Functionally, Akt supports cell survival by inhibiting apoptosis, achieved through phosphorylation and inactivation of multiple pro-apoptotic targets, including Bad, forkhead transcription factors, c-Raf, and caspase-9. Akt also drives cell growth by directly phosphorylating mTOR within a rapamycin-sensitive mTOR–raptor complex and by inactivating tuberin (TSC2), a negative regulator of mTOR, thereby sustaining mTOR pathway activation.

Background

Akt—also known as protein kinase B (PKB) or Rac—is a pivotal regulator of cell survival and apoptosis. It is activated by insulin as well as various growth and survival factors via a PI3 kinase–dependent pathway that is sensitive to wortmannin. Of its three isoforms, Akt1, Akt2, and Akt3, Akt1 has been studied most extensively in the contexts of cancer and metabolism. Akt1 is a central effector in the phosphatidylinositol 3,4,5-triphosphate (PIP3) signaling cascade and belongs to the AGC family of over 60 protein kinases, many of which are regulated through phosphorylation at both the activation loop and the C-terminal tail. In some AGC kinases, the C-terminal phosphorylation site is naturally replaced by an acidic aspartate, and for Akt1, the S473D substitution mimics the phosphorylated Ser473 state. In addition to the canonical Ser473 phosphorylation, Akt1 can also be phosphorylated at Ser477 and Thr479 by Cdk2/cyclinA2 within the nucleus. These non-canonical C-terminal modifications have been proposed to activate Akt1 in the nuclear compartment and promote cell-cycle progression. Mechanistic studies using Asp/Glu substitutions and peptide complementation suggest these modifications activate Akt1 through a process similar to Ser473 phosphorylation. Functionally, Akt supports cell survival by inhibiting apoptosis, achieved through phosphorylation and inactivation of multiple pro-apoptotic targets, including Bad, forkhead transcription factors, c-Raf, and caspase-9. Akt also drives cell growth by directly phosphorylating mTOR within a rapamycin-sensitive mTOR–raptor complex and by inactivating tuberin (TSC2), a negative regulator of mTOR, thereby sustaining mTOR pathway activation.

References
https://pubmed.ncbi.nlm.nih.gov/30078705/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3
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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%