Phosphoproteins FAQ - Orders/Support

Phosphoproteins

How are your new Phospho-Akt (Ser473) and Total Akt Kits different than your existing kits?

Our new Phospho-Akt (Ser473) Kit (catalog#K150MND) and Total Akt Kit (catalog#K150MOD) provide many advantages for the absolute quantification of Akt1 in biological samples. The new assays have been developed with recombinant calibrators and highly characterized critical reagents for greater sensitivity and better reproducibility. The recombinant calibrator included in the kits can be used to generate a standard curve to quantitate phosphorylated Akt at serine 473 or total Akt levels in human, mouse, rat, and non-human primate cell lysates as well as tissue lysate samples. Read all of our Akt product FAQs for more details.

Q:	What standards are used to calibrate the assays?
A:	Most assays are developed using Whole Cell Lysate Sets. The Phospho-Akt (Ser473) Kit for new and Total Akt Kit are provided with recombinant calibrators which have been in vitro phosphorylated.

Q:	What is the formulation of MSD^® Tris Lysis Buffer?
A:	150 mM NaCl 20 mM Tris, pH 7.5 1 mM EDTA 1 mM EGTA 1% Triton X-100 Complete lysis buffer contains protease and phosphatase inhibitors which are available in the MSD Inhibitor Pack.

Q:	Can I denature the cell lysates?
A:	Yes, ionic detergents or urea can be used to denature proteins, however these reagents must be diluted before adding the sample to the MSD plate in order to avoid denaturing antibodies. Ionic detergents should be diluted to 0.1% or less and urea should be diluted to 1 M or less.

How do I measure percent phosphorylation?

Perform a lysate titration to establish the linear range of the assay. For samples in the linear range, multiply the signal for the "phospho" spot by 2 and divide by the sum of the signal from the "phospho" and "total" spots, then multiply that value by 100 ((2xphospho / phospho + total) * 100). Each phosphoprotein assay kit product insert contains a detailed explanation of the percent phosphorylation with specific examples.

Q:	What other lysis buffers have been used with MSD assays?
A:	Various modified RIPA buffers with low concentrations of ionic detergents and urea-based lysis buffers have successfully been used in the assays. The MSD Lysis Buffer is optimal for many assays; however any buffer that is compatible with traditional ELISA should be suitable for use in MSD assays.

Q:	Why do I not see the same dynamic range as I see with cytokine assays?
A:	Non-specific binding of antibodies, especially phospho-specific antibodies, limits the dynamic range of the assays due to the binding of unrelated, abundant phosphoproteins in the lysate.

Q:	When I add more lysate to increase assay signals, why do the signals continue to decrease?
A:	High concentration of proteins in the sample can inhibit antibody binding, especially with phospho-specific antibodies.

Q:	Should I avoid any reagents in my lysis buffer?
A:	High concentrations of ionic detergents (> 0.1%), urea (> 1 M), and other protein denaturants should be avoided. These constituents can be used if they are subsequently diluted prior to adding the sample to the MSD plate (so as to prevent denaturation of the antibodies).

Q:	Is a phospho-specific antibody always used to capture the phosphoprotein?
A:	The phospho-specific antibody may be the capture or the detection antibody with one exception. In duplex assays that measure the phospho and total populations of one protein, the "phospho" spot must use the anti-phospho-specific antibody as the capture.

Q:	What parameters can I optimize for my particular sample?
A:	End users can vary 1) the concentration of detection antibody used, 2) the time that the lysate is incubated in the plate and 3) the use of off-the-shelf blockers in the lysis buffer and antibody dilution buffer to optimize the assay for a particular sample.

Q:	Can the assays be used with tissue lysates?
A:	Yes, brain tissue homogenates, liver homogenates, homogenates from Matrigel plugs, and various other sample types have been used in MSD Phosphoprotein Assays.

Q:	How are MSD assays validated?
A:	A cell model that exhibits regulation of a phosphoprotein of interest is selected from the scientific literature and the cell line is established in MSD labs. Antibodies are selected and an assay is released once the MSD sandwich assay agrees with western-blot analysis of the same samples.

Q:	Can I develop my own phosphoprotein assays?
A:	Yes, immobilizing antibodies on bare plates and labeling detection antibodies is very straightforward. Protocols and methods are readily available on the MSD website. Detailed information is available in the MULTI-ARRAY^® 96-well Plates and MSD SULFO-TAG™ NHS-Ester application notes.

Q:	How can I control cross-reactivity in multiplex assays?
A:	MSD has several blockers available that can be added to lysis buffer or antibody diluent to control cross-reactivity. The most common cross-reactivity remedies are the use of dried milk in lysis buffer and the use of non-specific IgGs in antibody diluent.

How do I know what amount of lysate to use in MSD Phosphoprotein Kits?

The amount of lysate to use in the kit will depend greatly on the abundance of the protein of interest in the particular cell type or tissue type to be studied. The minimum amount that can be used will depend on the abundance of the phosphoprotein and the percentage that is phosphorylated within the sample of interest. 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 assay signals across treatments to identify a common lysate amount for subsequent experiments.

Q:	What volume of lysate should I use?
A:	A minimum of 25 µL should be placed in each well to ensure adequate coverage of each electrode. Adding more lysate to the well will provide more analyte for capture, but few assays benefit from greater than 75 µL in the well.

Q:	Is phenylmethanesulfonyl fluoride (PMSF) a required component of the lysis buffer?
A:	No. Inclusion may be cell-line dependent. You may optimize the lysis buffer for your application.

Q:	I am struggling to solubilize the PMSF. Can you offer any suggestions?
A:	Try these: Bring the PMSF and additional lysis buffer components to room temperature before adding the PMSF. (PMSF is not soluble in cold solutions.) Add PMSF in 4 x 10 µL increments, mixing well. If precipitate is observed after mixing well, then we recommend preparing a new solution.

Q:	Can I use AEBSF as a substitute for PMSF?
A:	AEBSF is a component of the protease inhibitor solution used in preparation of MSD complete lysis buffer. Inclusion of PMSF provides additional serine protease inhibition for some cell lines. You may eliminate PMSF depending on your cell-line and optimize the lysis buffer for your application. If you prefer not to use PMSF, try optimizing lysis buffer conditions by PMSF titration.

Q:	How long can I store 1X Tris Wash Buffer at room temperature?
A:	1X Tris Wash Buffer may be stored at room temperature until the expiration date listed on the 10X bottle provided the buffer is prepared under sterile conditions.