Phosphoproteins

Q: What standards are used to calibrate the assays? A: The assays are developed using positive and negative control Whole Cell Lysate Sets. Q: What is the MSD® Lysis Buffer formulation? 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; more detailed information is available in the MSD Lysate Preparation Protocol. 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. Q: How do I measure percent phosphorylation? A: 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. Q: What amount (micrograms) of lysate should I use? A: The performance of the assay will depend greatly on the abundance of the protein of interest in the particular cell type or tissue type to be studied. The assays seldom benefit from more than 20 µg of lysate in the well. 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. The most robust assays can be performed with less than 1 µg of total protein. 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.