Anti-AKT1,2,3 Antibody Picoband™

Product Name

Anti-AKT1,2,3 Antibody Picoband™

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SKU/Catalog Number

A00024-2

Size

100 μg/vial

Form

Lyophilized

Description

Boster Bio Anti-AKT1,2,3 Antibody Picoband™ catalog # A00024-2. Tested in ELISA, Flow Cytometry, IF, ICC, WB applications. This antibody reacts with Human, Mouse, Rat.

Storage & Handling

At -20°C for one year from date of receipt. After reconstitution, at 4°C for one month. It can also be aliquotted and stored frozen at -20°C for six months. Avoid repeated freezing and thawing.

Cite This Product

Anti-AKT1,2,3 Antibody Picoband™ (Boster Biological Technology, Pleasanton CA, USA, Catalog # A00024-2)

Host

Rabbit

Contents

Each vial contains 4 mg Trehalose, 0.9 mg NaCl, 0.2 mg Na2HPO4.

Clonality

Polyclonal

Isotype

Rabbit IgG

Immunogen

E.coli-derived human AKT1,2,3 recombinant protein (Position: E17-A477).

*Blocking peptide can be purchased. Costs vary based on immunogen length. Contact us for pricing.

Cross-reactivity

No cross-reactivity with other proteins.

Reactive Species

A00024-2 is reactive to in Human, Mouse, Rat

Applications

A00024-2 is guaranteed for ELISA, Flow Cytometry, IF, ICC, WB Boster Guarantee

Observed Molecular Weight

60 kDa

Calculated molecular weight

134277 MW

Background of

RAC-alpha serine/threonine-protein kinase is an enzyme that in humans is encoded by the AKT1 gene. This gene encodes one of the three members of the human AKT serine-threonine protein kinase family which are often referred to as protein kinase B alpha, beta, and gamma. These highly similar AKT proteins all have an N-terminal pleckstrin homology domain, a serine/threonine-specific kinase domain and a C-terminal regulatory domain. These proteins are phosphorylated by phosphoinositide 3-kinase (PI3K). AKT/PI3K forms a key component of many signalling pathways that involve the binding of membrane-bound ligands such as receptor tyrosine kinases, G-protein coupled receptors, and integrin-linked kinase. These AKT proteins therefore regulate a wide variety of cellular functions including cell proliferation, survival, metabolism, and angiogenesis in both normal and malignant cells. AKT proteins are recruited to the cell membrane by phosphatidylinositol 3,4,5-trisphosphate (PIP3) after phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) by PI3K. Subsequent phosphorylation of both threonine residue 308 and serine residue 473 is required for full activation of the AKT1 protein encoded by this gene. Phosphorylation of additional residues also occurs, for example, in response to insulin growth factor-1 and epidermal growth factor. Protein phosphatases act as negative regulators of AKT proteins by dephosphorylating AKT or PIP3. The PI3K/AKT signalling pathway is crucial for tumor cell survival. Survival factors can suppress apoptosis in a transcription-independent manner by activating AKT1 which then phosphorylates and inactivates components of the apoptotic machinery. AKT proteins also participate in the mammalian target of rapamycin (mTOR) signalling pathway which controls the assembly of the eukaryotic translation initiation factor 4F (eIF4E) complex and this pathway, in addition to responding to extracellular signals from growth factors and cytokines, is disregulated in many cancers. Mutations in this gene are associated with multiple types of cancer and excessive tissue growth including Proteus syndrome and Cowden syndrome 6, and breast, colorectal, and ovarian cancers. Multiple alternatively spliced transcript variants have been found for this gene.

Antibody Validation

Boster validates all antibodies on WB, IHC, ICC, Immunofluorescence, and ELISA with known positive control and negative samples to ensure specificity and high affinity, including thorough antibody incubations.

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Reconsitution

Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.

Assay Dilutions Recommendation

The recommendations below provide a starting point for assay optimization. The actual working concentration varies and should be decided by the user.

Western blot, 0.25-0.5 μg/ml, Human, Mouse, Rat
Immunocytochemistry/Immunofluorescence, 5 μg/ml, Human
Flow Cytometry, 1-3 μg/1x10⁶ cells, Human
Direct ELISA, 0.1-0.5 μg/ml, Human

Validation Images & Assay Conditions

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Figure 1. Western blot analysis of AKT1,2,3 using anti-AKT1,2,3 antibody (A00024-2).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human A549 whole cell lysates,
Lane 2: human Hela whole cell lysates,
Lane 3: human HepG2 whole cell lysates,
Lane 4: human Raji whole cell lysates,
Lane 5: rat brain tissue lysates,
Lane 6: rat PC-12 whole cell lysates,
Lane 7: mouse brain tissue lysates,
Lane 8: mouse RAW264.7 whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-AKT1,2,3 antigen affinity purified polyclonal antibody (Catalog # A00024-2) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for AKT1,2,3 at approximately 60 kDa. The expected band size for AKT1,2,3 is at 60 kDa.

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Figure 2. IF analysis of AKT1,2,3 using anti-AKT1,2,3 antibody (A00024-2).
AKT1,2,3 was detected in an immunocytochemical section of MCF-7 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-AKT1,2,3 Antibody (A00024-2) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

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Figure 3. Flow Cytometry analysis of Hela cells using anti-AKT1,2,3 antibody (A00024-2).
Overlay histogram showing Hela cells stained with A00024-2 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-AKT1,2,3 Antibody (A00024-2, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

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