Figure 1: AKT1 target protein structure.

Introduction to AKT1 target

Protein function

• AKT1 (serine/threonine kinase AKT) is also known as protein kinase B (PKB). AKT1 is activated by insulin and various growth and survival factors, and plays a key role in regulating cell growth, division, and apoptosis inhibition, among other processes.
• The AKT family has three members, with AKT1 and AKT2 being more widely distributed. As a key protein in the PI3K/AKT/mTOR signaling pathway, AKT1 can prevent apoptosis and promote cell survival, and is considered a major factor in many types of cancer.

Protein expression

• AKT1 is expressed in various  cell types or tissues,  with low tissue specificity.

Protein localization

• AKT1 is mainly localized in the cytoplasm and nucleus of cells. After activation, AKT1 translocates to the nucleus.

Figure 2: AKT1 ICC experimental result image, Anti-AKT1 antibody [1F7E10] (ab233755)

Sample name: HepG2 cell line.

Green: AKT1, Red: Actin, Blue: DRAQ5 DNA dye

Isoforms & Post-translational modifications

• Human (P31749): Isoform 1: 56 kDa; Isoform 2: 48 kDa (predicted)
• Mouse (P31750): 56 kDa (predicted)
• Rat (P47196): 56 kDa (predicted)
• Various post-translational modifications including glycosylation, phosphorylation, ubiquitination, and acetylation.

AKT1 experimental tips

Precautions:

• The expression of AKT1 varies in different cells or tissues. Weak or no signal may be observed in same samples. Please select appropriate samples and use positive controls to confirm that the experimental procedure isworking properly.
• Stimulation(e.g. with Calyculin A reagent) is typically required for the detection of phosphorylated AKT1.
• Please confirm the expression level of total AKT1 When detecting phosphorylated AKT1.
• Add appropriate phosphatase inhibitors.
• Please use freshly prepared lysates.

Positive controls:

• AKT1: MCF7, HeLa cell lines
• AKT1 (phospho S473): LNCaP cell line treated with 0.1 μM Calyculin A for 30 minutes.

Example of results:

Figure 3: Recombinant Anti-AKT1 (phospho S473) antibody [EP2109Y] (ab81283)

Lane 1: NIH/3T3 treated with 100ng/ml PDGF for 1 hour whole cell lysates
Lane 2: NIH/3T3 whole cell lysates

Predicted band size: 56 kDa
Observed band size: 56 kDa

Figure 4: Recombinant Anti-AKT1 (phospho S473) antibody [EP2109Y] (ab81283)

Lane 1: LNCaP whole cell lysates
Lane 2: LNCaP treated with 0.1 μM Calyculin A for 30 minutes whole cell lysates
Lane 3: A549 whole cell lysates
Lane 4: MCF7 whole cell lysates
Lane 5: HepG2 whole cell lysates
Lane 6: HUVEC whole cell lysates
Lane 7: C2C12 whole cell lysates
Lane 8: Mouse brain tissue lysates
Lane 9: Rat heart tissue lysates
Predicted band size: 56 kDa

The basal expression level of AKT1 (phospho S473) varies in different cell lines reported by PMID: 19372546. But to detect clear signal, treatment is strongly recommended when using this antibody.

Figure 5: Recombinant Anti-AKT1 + AKT2 + AKT3 antibody [EPR16798] (ab179463)

Lane 1: MCF7 whole cell lysates
Lane 2: HeLa whole cell lysates
Lane 3: Hep G2 whole cell lysates
Lane 4: A549 whole cell lysates
Predicted band size: 56 kDa
Observed band size: 56 kDa

*The image shows the detection results for AKT1, AKT2, and AKT3.

Key control points

Please pay attention to following general tips to help ensure the best results:

Sample preparation:

1. Add adequate protease inhibitor Cocktail to avoid target protein dagradation.
2. Keep samples on ice during the sample preparation process.
3. Perform a Bradford assay, a Lowry assay or a bicinchoninic acid (BCA) assay to determine the protein concentration.

Electrophoresis:

1. Load at least 20μg total protein per lane.

Transferring:

1. Use Ponceau S staining to determine if the transfer is successful.

Blocking:

1. Choose the appropriate blocking buffer according to the datasheet.
2. Antibody incubation:
3. Determine the optimal working dilution of the antibody according to the datasheet.

References

1. Brendan D Manning, Alex Toker. AKT/PKB Signaling: Navigating the Network. Cell. 2017 Apr 20;169(3):381-405. doi: 10.1016/j.cell.2017.04.001.
2. Kyung H Yi, Josh Lauring. Recurrent AKT mutations in human cancers: functional consequences and effects on drug sensitivity. Oncotarget. 2016 Jan 26;7(4):4241-51. doi: 10.18632/oncotarget.6648.
3. Mireia Guerau-de-Arellano, Zayda L Piedra-Quintero, Philip N Tsichlis. Akt isoforms in the immune system. Front Immunol. 2022 Aug 23;13:990874. doi: 10.3389/fimmu.2022.990874. eCollection 2022.
4. J Pascual, N C Turner. Targeting the PI3-kinase pathway in triple-negative breast cancer. Ann Oncol. 2019 Jul 1;30(7):1051-1060. doi: 10.1093/annonc/mdz133.