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FAQs for Phospho-Akt (Ser473) Kit and Total Akt Kits (Product info)

  1. Do the new total Akt and phospho-Akt (Ser473) assays use the same antibody pairs as the old assays?

    No. The new assays use different antibodies. The old assays used antibodies from commercial vendors, while the new assays use MSD mouse monoclonal antibodies that have been well characterized through MSD’s critical reagents program. Both the old and new antibodies have been confirmed to bind only to Akt1. No significant cross-reactivity with Akt2 or Akt3 has been observed.     

  1. How do the new Phospho-Akt (Ser473) Kits and Total Akt Kits compare to the previous kits in terms of performance?

    The new kits are more sensitive and have been tested in human, mouse, rat, and non-human primate cell lysates as well as tissue lysate samples (tumor xenografts). The new kits provide quantitative measurements of intracellular proteins. A full-length recombinant calibrator is included in the kits that can be used to generate a standard curve to quantitate phospho Akt or total Akt levels in test samples.
  1. What research species have been tested with Phospho-Akt (Ser473) Kits and Total Akt Kits kits?

    We have confirmed that the Phospho-Akt (Ser473) Kit and Total Akt Kit cross-react with human, mouse, rat, and non-human primate lysates.
  2. Are cell or tissue lysis buffers included in the Phospho-Akt (Ser473) Kits and Total Akt Kits?

    MSD Tris Lysis Buffer and Inhibitor Pack are available for separate purchase from MSD and can be used for cell lysate or tissue lysate preparations. If you wish to prepare lysis buffer at the bench, the components of the MSD Tris Lysis Buffer are listed in Appendix A of each kit’s product insert.
  3. What is the difference between Diluent 39 and Tris Lysis Buffer?

    Diluent 39 is MSD Tris Lysis Buffer supplemented with carrier protein.
  4. I ordered the Phospho-Akt (Ser473) Kit, yet the plate is labeled as a 96-well 4-spot Akt Plate. Did I receive the incorrect plate?

    You have received the correct plate. Both kits use the same plate because both assays use the same capture antibody. The difference between kits is the detection antibody. The Total Akt Kit contains detection antibody that binds both phosphorylated and non-phosphorylated Akt, while the Phospho-Akt (Ser473) Kit will bind only the Akt that has been phosphorylated at serine 473.
  5. I have a vial of calibrator that is labeled Phospho-Akt (Ser473) Calibrator and a vial that is labeled Akt Calibrator. Are they the same?

    Yes, they are from different manufacturing lots but both are full length recombinant human Akt1 protein expressed in baculovirus and phosphorylated in vitro using phosphoinositide-dependent protein kinase-1 (PDK1) and MAP kinase-activated protein kinase 2 (MAPKAPK2). For the actual calibrator concentration, refer to the Certificate of Analysis (COA) supplied with the kit.
  6. Can I order additional calibrator for the Phospho-Akt (Ser473) Kits and Total Akt Kits?

    Yes, the catalog number is C00MN-2.
  7. Can I order additional detection antibody for the Phospho-Akt (Ser473) Kits and Total Akt Kits?

    Yes, the catalog numbers are:

    D20MN-2              Phospho-Akt (Ser473) Antibody, 1-plate vial
    D20MN-3              Phospho-Akt (Ser473) Antibody, 5-plate vial
    D20MO-2              Total Akt Antibody, 1-plate vial
    D20MO-3              Total Akt Antibody, 5-plate vial

  1. Are the NIH3T3, Rat L6, and COS7 cell lysate sets featured in the Phospho-Akt (Ser473) Kits and Total Akt product insert available for purchase?

    No, but they are commercially available cell lines and the compounds used for treatment are also commercially available. Details regarding the treatment used to generate each cell lysate are provided in the product inserts.

  1. What are the differences between Phospho-Akt (Ser473) Whole Cell Lysate Set and the Akt Control Pack?

    MSD whole cell lysate sets provide qualitative positive and negative controls, specifically lysates of growing Jurkat cells (positive) and cells that have been treated with LY294002 to inhibit phosphorylation of Akt (negative). The lysates are diluted prior to use.

    The Akt Control Pack includes lysates of growing Jurkat cells in two concentrations that span the dynamic range of the assays. The lysates are provided “ready to use”. We provide lot-specific concentrations for each control in the product COA. These lysates are stable through at least one additional freeze-thaw cycle.

  1. Can the Phospho-Akt (Ser473) Whole Cell Lysate Set (catalog # C11CA-1) be used with the new Akt kits?

    Yes. However, expected concentrations for phospho-Akt (Ser473) and total Akt are not provided for the MSD whole cell lysate set.

  1. Is there a difference between the calibrator used in the Total Akt and the Phospho-Akt (Ser473) Kits?

    No, the Akt calibrator is identical. This allows the end user to use one calibrator in any of the Akt-related singleplex assays.

  1. What are the concentrations of the Akt calibrator stocks?

    The concentrations of the Akt calibrator stock is provided in the kit lot COA . For phospho-Akt, the COA will list the calibrator units in UP473/well. For total Akt, the COA will list the calibrator units in pg/well.

  1. The calibrator stock is provided in Units/well for phospho-Akt and pg/well for total Akt.  Is it feasible to convert the concentration into Units/mL for phospho-Akt and pg/mL for total Akt?

    Yes, simply divide the concentration of the calibrator stock by the volume of calibrator which will be added to the assay well.  For example, 1000 Units/well of phospho-Akt divided by 0.025mL equates to 40,000 Units/mL.  25,000 pg/well of total Akt divided by 0.025mL equates to 1,000,000 pg/mL or 1µg/mL.

  1. How is phospho-Akt (Ser473) concentration for the calibrator assigned?

    Units are assigned based on an internal reference material.

    One UP473 equates to the phosphorylation of 100 pg of the reference lot of Akt1 recombinant protein in an in vitro kinase reaction.

  1. Can I use the Akt calibrator in the Phospho-Akt (Thr308) Whole Cell Lysate Kit.

    Yes, the calibrator is phosphorylated at Thr308, but the expected levels of Phospho-Akt (Thr308) are not provided for the Akt calibrator.

  1. Can I use the Akt calibrator in the Phospho(Ser473)/Total Akt Whole Cell Lysate Kit.

    We do not recommend using the Akt calibrator in the duplex kit.
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