AKT1 Overexpression Lysate

Novus Biologicals | Catalog # NBP2-04280

Novus Biologicals
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Key Product Details

Species

Human

Applications

Western Blot

Product Summary for AKT1 Overexpression Lysate

AKT1 Transient Overexpression Lysate


Expression Host: HEK293T

Plasmid: RC220257

Accession#: NM_005163

Protein Tag: C-MYC/DDK

You will receive 1 vial of lysate (100ug), 1 vial of empty vector negative control (100ug), and 1 vial of 2xSDS sample buffer (250ul). Each vial of cell lysate contains 100ug of total protein (at 1 mg/ml). The 2xSDS Sample Buffer consists of 4% SDS, 125mM Tris-HCl pH6.8, 10% Glycerol, 0.002% Bromophenol blue, 100mM DTT.

Product Specifications

Application Notes

This product is intended for use as a positive control in Western Blot. Overexpression of the target protein was confirmed using an antibody to DDK (FLAG) epitope tag (NBP1-71705) present on the protein construct.

Each vial of cell lysate contains 100ug of total protein which should be sufficient for 20-50 reactions. Depending on over-expression level, antibody affinity and detection system, some lysates can go as low as 0.1 ug per load. We recommend starting with 5ug of cell lysate. Add an equal amount of cell lysate and 2X SDS Sample buffer and boil the SDS samples for 10 minutes before loading.

Theoretical MW

55.5 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Type

Overexpression

Scientific Data Images for AKT1 Overexpression Lysate

Western Blot: AKT1 Overexpression Lysate [NBP2-04280]

Western Blot: AKT1 Overexpression Lysate [NBP2-04280]

Western Blot: AKT1 Overexpression Lysate (Adult Normal) [NBP2-04280] Left-Empty vector transfected control cell lysate (HEK293 cell lysate); Right -Over-expression Lysate for AKT1.

Formulation, Preparation, and Storage

Formulation

RIPA buffer

Concentration

The exact concentration of the protein of interest cannot be determined for overexpression lysates. Please contact technical support for more information.

Shipping

The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.

Storage

Store at -80C. Avoid freeze-thaw cycles.

Background: Akt1

AKT (also known as protein kinase B (PKB) and RAC (related to A and C kinases)) is a critical intracellular serine/threonine kinase that translates signals from extracellular stimuli including growth factors, cytokines and neurotransmitters (1). AKT signaling plays critical roles in cell growth, proliferation, survival and differentiation (1). It is also involved in organogenesis, angiogenesis and metabolism. Three mammalian AKT isoforms have been identified. The AKT pathway can be activated by any of the three members who share a high level of protein homology but are independently encoded by AKT1 (PKB alpha; 14q32.32), AKT2 (PKB beta; 19q13.2), or AKT3 (PKB gamma; 1q44) (1, 2). Each AKT family member contains an N-terminal pleckstrin homology (PH) domain, a central kinase domain, and a C-terminal regulatory domain. AKT mediates many of the downstream events of phosphatidylinositol 3-kinase (PI3-K), a lipid kinase activated by growth factors, cytokines and insulin. PI3-K recruits AKT to the membrane, where it is activated by PDK1 phosphorylation. AKT has two main phosphorylation sites (Ser473 and Thr308, predicted molecular weight 56 kDa) (3, 4). Once phosphorylated, AKT dissociates from the membrane and phosphorylates targets in the cytoplasm and the cell nucleus including mammalian target of rapamycin (mTOR).

The main function of AKT is to control inhibition of apoptosis and promote cell proliferation. Survival factors can activate AKT Ser473 and Thr308 phosphorylation sites in a transcription-independent manner, resulting in the inactivation of apoptotic signaling transduction through the tumor suppressor PTEN, an antagonist to PI3-K (5). PTEN exerts enzymatic activity as a phosphatidylinositol-3,4,5-trisphosphate (PIP3) phosphatase, opposing PI3K activity by decreasing availability of PIP3 to proliferating cells, leading to overexpression and inappropriate activation of AKT noted in many types of cancer.

AKT1 function has been linked to overall physiological growth and function (2). AKT1 has been correlated with proteus syndrome, a rare disorder characterized by overgrowth of various tissues caused by a mosaic variant in the AKT1 gene in humans.

AKT2 is strongly correlated with Type II diabetes, including phenotypes of insulin resistance, hyperglycemia and atherosclerosis (2, 6).

The function of AKT3 is specifically associated to brain development, where disruptions to AKT3 are correlated with microcephaly, hemimegalencephaly, megalencephaly and intellectual disabilities (2).

References

1. Ersahin, T., Tuncbag, N., & Cetin-Atalay, R. (2015). The PI3K/AKT/mTOR interactive pathway. Mol Biosyst, 11(7), 1946-1954. doi:10.1039/c5mb00101c

2. Cohen, M. M., Jr. (2013). The AKT genes and their roles in various disorders. Am J Med Genet A, 161a(12), 2931-2937. doi:10.1002/ajmg.a.36101

3. Georgescu, M. M. (2010). PTEN Tumor Suppressor Network in PI3K-Akt Pathway Control. Genes Cancer, 1(12), 1170-1177. doi:10.1177/1947601911407325

4. Mishra, P., Paital, B., Jena, S., Swain, S. S., Kumar, S., Yadav, M. K.,... Samanta, L. (2019). Possible activation of NRF2 by Vitamin E/Curcumin against altered thyroid hormone induced oxidative stress via NFkB/AKT/mTOR/KEAP1 signalling in rat heart. Sci Rep, 9(1), 7408. doi:10.1038/s41598-019-43320-5

5. Wedel, S., Hudak, L., Seibel, J. M., Juengel, E., Oppermann, E., Haferkamp, A., & Blaheta, R. A. (2011). Critical analysis of simultaneous blockage of histone deacetylase and multiple receptor tyrosine kinase in the treatment of prostate cancer. Prostate, 71(7), 722-735. doi:10.1002/pros.21288

6. Rotllan, N., Chamorro-Jorganes, A., Araldi, E., Wanschel, A. C., Aryal, B., Aranda, J. F.,... Fernandez-Hernando, C. (2015). Hematopoietic Akt2 deficiency attenuates the progression of atherosclerosis. Faseb j, 29(2), 597-610. doi:10.1096/fj.14-262097

Long Name

v-Akt Murine Thymoma Viral Oncogene Homolog 1

Alternate Names

PKB alpha, PRKBA, RAC-alpha

Gene Symbol

AKT1

Additional Akt1 Products

Product Documents for AKT1 Overexpression Lysate

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

Product Specific Notices for AKT1 Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48 hrs before collection. The cells were lysed in modified RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1X Proteinase inhibitor cocktail mix, 1 mM PMSF and 1 mM Na3PO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

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FAQs for AKT1 Overexpression Lysate

Showing  1 - 5 of 5 FAQs Showing All

  • Q: Do your HRP-conjugated antibodies contain sodium azide?

    A: No. None of our HRP-conjugated antibodies contain sodium azide as this agent inhibits the activity of HRP.

  • Q: How do I choose secondary antibodies to label the same cells when I have two primary antibodies from the same host?

    A: Use isotype-specific secondary antibodies if the primary antibodies are of different isotypes. You can also make direct conjugates of the primary antibodies by use of antibody labeling kits, dyes, or custom conjugations (please contact Technical Support for custom orders).

  • Q: I am looking for a antibody that recognizes human Akt1 but NOT Akt2 or 3, for Western blot analyses. I also want that antibody to recognize Akt1 regardless of its phosphorylated form.

    A: At the moment we do not have an AKT1 antibody that definitively does not react with either AKT2 or AKT3.

  • Q: What is the molecular weight of your antibodies?

    A: All IgG antibodies are approximately 150 kDa (each heavy chain is about 50 kDa and each light chain is about 25 kDa).

  • Q: Why are many of your antibodies formulated with sodium azide and BSA?

    A: Sodium azide is a preservative which is added to prevent bacterial growth. BSA is added as a protein stabilizer.

  • Q: Do your HRP-conjugated antibodies contain sodium azide?

    A: No. None of our HRP-conjugated antibodies contain sodium azide as this agent inhibits the activity of HRP.

  • Q: How do I choose secondary antibodies to label the same cells when I have two primary antibodies from the same host?

    A: Use isotype-specific secondary antibodies if the primary antibodies are of different isotypes. You can also make direct conjugates of the primary antibodies by use of antibody labeling kits, dyes, or custom conjugations (please contact Technical Support for custom orders).

  • Q: I am looking for a antibody that recognizes human Akt1 but NOT Akt2 or 3, for Western blot analyses. I also want that antibody to recognize Akt1 regardless of its phosphorylated form.

    A: At the moment we do not have an AKT1 antibody that definitively does not react with either AKT2 or AKT3.

  • Q: What is the molecular weight of your antibodies?

    A: All IgG antibodies are approximately 150 kDa (each heavy chain is about 50 kDa and each light chain is about 25 kDa).

  • Q: Why are many of your antibodies formulated with sodium azide and BSA?

    A: Sodium azide is a preservative which is added to prevent bacterial growth. BSA is added as a protein stabilizer.

  • Q: Do your HRP-conjugated antibodies contain sodium azide?

    A: No. None of our HRP-conjugated antibodies contain sodium azide as this agent inhibits the activity of HRP.

  • Q: How do I choose secondary antibodies to label the same cells when I have two primary antibodies from the same host?

    A: Use isotype-specific secondary antibodies if the primary antibodies are of different isotypes. You can also make direct conjugates of the primary antibodies by use of antibody labeling kits, dyes, or custom conjugations (please contact Technical Support for custom orders).

  • Q: I am looking for a antibody that recognizes human Akt1 but NOT Akt2 or 3, for Western blot analyses. I also want that antibody to recognize Akt1 regardless of its phosphorylated form.

    A: At the moment we do not have an AKT1 antibody that definitively does not react with either AKT2 or AKT3.

  • Q: What is the molecular weight of your antibodies?

    A: All IgG antibodies are approximately 150 kDa (each heavy chain is about 50 kDa and each light chain is about 25 kDa).

  • Q: Why are many of your antibodies formulated with sodium azide and BSA?

    A: Sodium azide is a preservative which is added to prevent bacterial growth. BSA is added as a protein stabilizer.

  • Q: Do your HRP-conjugated antibodies contain sodium azide?

    A: No. None of our HRP-conjugated antibodies contain sodium azide as this agent inhibits the activity of HRP.

  • Q: How do I choose secondary antibodies to label the same cells when I have two primary antibodies from the same host?

    A: Use isotype-specific secondary antibodies if the primary antibodies are of different isotypes. You can also make direct conjugates of the primary antibodies by use of antibody labeling kits, dyes, or custom conjugations (please contact Technical Support for custom orders).

  • Q: I am looking for a antibody that recognizes human Akt1 but NOT Akt2 or 3, for Western blot analyses. I also want that antibody to recognize Akt1 regardless of its phosphorylated form.

    A: At the moment we do not have an AKT1 antibody that definitively does not react with either AKT2 or AKT3.

  • Q: What is the molecular weight of your antibodies?

    A: All IgG antibodies are approximately 150 kDa (each heavy chain is about 50 kDa and each light chain is about 25 kDa).

  • Q: Why are many of your antibodies formulated with sodium azide and BSA?

    A: Sodium azide is a preservative which is added to prevent bacterial growth. BSA is added as a protein stabilizer.

  • Q: Do your HRP-conjugated antibodies contain sodium azide?

    A: No. None of our HRP-conjugated antibodies contain sodium azide as this agent inhibits the activity of HRP.

  • Q: How do I choose secondary antibodies to label the same cells when I have two primary antibodies from the same host?

    A: Use isotype-specific secondary antibodies if the primary antibodies are of different isotypes. You can also make direct conjugates of the primary antibodies by use of antibody labeling kits, dyes, or custom conjugations (please contact Technical Support for custom orders).

  • Q: I am looking for a antibody that recognizes human Akt1 but NOT Akt2 or 3, for Western blot analyses. I also want that antibody to recognize Akt1 regardless of its phosphorylated form.

    A: At the moment we do not have an AKT1 antibody that definitively does not react with either AKT2 or AKT3.

  • Q: What is the molecular weight of your antibodies?

    A: All IgG antibodies are approximately 150 kDa (each heavy chain is about 50 kDa and each light chain is about 25 kDa).

  • Q: Why are many of your antibodies formulated with sodium azide and BSA?

    A: Sodium azide is a preservative which is added to prevent bacterial growth. BSA is added as a protein stabilizer.

Showing  1 - 5 of 5 FAQs Showing All
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