Note: In case follow-up experiments are needed, it is strongly recommended to sub-aliquot all samples after preparation to minimize cytokine degradation from multiple freeze-thaw cycles.
We recommend preparing serum-free or low-serum medium samples, as serum tends to contain cytokines which may produce significant background signals. If it is necessary to test serum containing medium, we recommend also running an uncultured media blank to assess baseline signals. This baseline can then be subtracted from the cultured media sample data.
*The optimal number of seeded cells varies from one cell type to another and may need to be empirically determined.
Cell or tissue lysates for use with RayBio® Antibody Arrays, Glycobiology Arrays, Protein Arrays and ELISA kits can be prepared using most conventional methods, e.g. homogenization of cell or tissue in RayBio® Lysis Buffer (AA-LYS or EL-LYSIS). You may also use your own lysis buffer, such as RIPA or other formulations optimized for immunoprecipitation.
We strongly recommend adding a protease inhibitor cocktail to the lysis buffer prior to homogenization. You may use RayBiotech Protease Inhibitor Cocktail (AA-PI), RayBiotech Phosphatase Inhibitor Cocktail Set I (AA-PHI-I) or Phosphatase Inhibitor Cocktail Set II (AA-PHI-II), or the RayBiotech Phosphatase / Protease Inhibitor Set (AA-PIPHI). Alternatively, you can purchase inhibitor cocktails from another manufacturer and follow their specifications. Most general biochemical supply companies including Roche, Sigma-Aldrich, Pierce, and Calbiochem stock a wide variety of these products. Since susceptibility to proteolytic cleavage and the type of proteases present in the lysate vary, we do not recommend a specific product. Instead, your choice of which combination of protease inhibitors to use should be based upon a literature search for your protein(s) of interest and/or tissue or cell type. Phosphatase inhibitors may be used but are not necessary unless the antibodies used in the kit specifically recognize phosphorylated forms of the protein.
Choices of the method for lysis and homogenization include glass-bead “smash,” douncing, freezethaw, sonication and crushing frozen tissue with a mortar and pestle, or even a combination of these. There is no best method for all sample types; your choice of method should be made following a brief search of the literature to see how samples similar to yours have been prepared in previous investigations.
After homogenization, centrifuge the lysates to remove cell/tissue debris (5 min @ 10,000 x g or 10 min @ 5,000 x g) and save the supernatant. Unless testing fresh, lysates should be frozen as soon as possible and stored at -20°C (or -80°C, if possible). Centrifuge them again before incubating with anyimmunoassay. Next, determine the protein concentration of your lysates using a total protein assay not inhibited by detergents (such as the Bicinchoninic acid (BCA) assay) and normalize the volume of each sample used to deliver the same amount of total protein for each assay.
Note: The Bradford assay is not recommended as it can be inhibited by the presence of detergents.
Since different cells and tissues may contain different amounts of protein, as starting point, we suggest using 500 µL of lysis buffer per 1x106 cells or 10 mg tissue. You may have to adjust this based upon your results. Your target total protein concentration of the homogenate should be at least 1,000 µg/mL, but 2,000 µg/mL or more would be better.