1. I would like to run my samples for the Phospho-Akt (Ser473) Kits and Total Akt Kits undiluted. They were prepared in a different lysis buffer than the one MSD offers. Should I still use the assay diluent from the kit to prepare my calibrator curve?

Yes. The calibrator curve should be prepared in assay diluent from the kit even if your sample lysis buffer differs.

Since most lysis buffers are compatible with MSD plates, you may supplement your lysis buffer with 1% w/v Blocker A for use in calibrator curve preparation if desired. High concentrations of denaturing reagents should be avoided. Keep SDS and other ionic detergents to a concentration of 0.1% or less in the sample applied to the well. Avoid reducing agents and chaotropes such as DTT >0.1mM and urea >0.1M. The effects of β-mercaptoethanol in the sample buffer have not been tested and should be evaluated by the end user where needed.

2. I am using tumor lysates as my sample matrix. Is an alternative lysis buffer recommended?

MSD Tris Lysis Buffer supplemented with the inhibitors from the Inhibitor Pack can be used to lyse tumor xenograft samples. You may need to use the phosphatase and protease inhibitor solutions at 2X final concentration or add additional inhibitors such as 2mM PMSF. Appropriate lysis conditions may be sample dependent, so you should optimize the lysis buffer for your application.

3. Will the Phospho-Akt (Ser473) Kits and Total Akt assay tolerate low concentrations of SDS in the cell lysis buffer?

Yes. When we tested a range of SDS concentrations, signals were retained with ≤0.1% SDS final concentration in the sample buffer.

4. Can I use assay diluent from the Phospho-Akt (Ser473) Kits and Total Akt Kits to lyse my cells?

Only in some circumstances. The presence of carrier protein in the assay diluent makes it unsuitable for use as a cell lysis buffer in situations where you need to quantify total lysate protein concentration. MSD Tris Lysis Buffer (catalog # R60TX-3) and Inhibitor Pack (catalog # R70AA-1) are available for separate purchase for preparation of lysate samples.

5. How do I know what amount of lysate to use in the Phospho-Akt (Ser473) Kits and Total Akt assay?

You may conduct a pilot study using a sample from each cell line and treatment. We suggest diluting study samples to a total protein concentration of 800 µg/mL (20 µg/well) and then serially diluting 2-fold to create a 7-point sample titration. Compare the resultant Akt concentrations across treatments to identify a common concentration for subsequent experiments. The selected concentration should allow quantification of all sample types within the linear range of the standard curve.

Partial plates can be used for pilot experiments. Guidelines and recommendations for using partial plates are provided in the kit product inserts.

6. Can I incubate samples overnight at 4C to maximize sensitivity?

Samples may be incubated overnight at 4C; however, for the samples we tested, we did not observe a significant improvement in sensitivity with overnight incubation.

7. Can I measure whole blood samples using the Phospho-Akt (Ser473) Kits and Total Akt Kits?

You can use lysed PMBCs from whole blood in the assays, but to use whole blood without PBMC extraction, you may need to further optimize the assay. Try using a modified or more concentrated lysis buffer.

8. Since the Phospho Akt (Ser473) Kit and the Total Akt Kit use the same plate and the protocol steps are aligned, can I simply purchase additional detection antibody and run half the plate for phospho-Akt (Ser473) and half the plate for total Akt?

This should be feasible, but be aware that we may not have tested your kit lot with the particular lot of additional detection antibody that you purchase.

9. Can I freeze the Phospho-Akt (Ser473) Kits and Total Akt assay diluent (Diluent 39 + protease & phosphatase inhibitors) for future use?

No. Fresh assay diluent should be made the day you run the assay.

10. Can I freeze calibrators for future use when using partial Phospho-Akt (Ser473) Kits and Total Akt plates?

Yes. The remaining Akt Calibrator (20X) is stable through at least two freeze-thaw cycles. However, diluted calibrators should be prepared for single use only and remainders should be discarded.

11. Has partial plate stability been tested for the Phospho-Akt (Ser473) Kits and Total Akt assays?

Partial plate stability has been evaluated; the product insert provides details regarding partial plate use and storage.