The Proteome Profiler™ arrays are considered semi-quantitative. Relative levels of protein concentration or phosphorylation are compared between samples by analyzing spot intensity on the array membrane.
The Array reference spots pixel densities are not proportional to the amount of protein loaded. The reference spots consist of unrelated biotinylated protein spotted at a minimum amount directly to the membranes for the purpose of confirming the kit detection system works and to assist in aligning the transparency overlay template with the array film or digital image.
The Array reference spots should not be used for normalization or quantitation primarily becuase the reference spots consist of unrelated biotinylated proteins spotted at minimal amount directly to the membranes, and the pixel densities of the reference spots are not proprotional to the amount of protein loaded on membranes. Experimental signals cannot be normalized to another set of sample data because each antibody may have a different affinity for its respective analyte, which means certain antibody combinations may detect protein at very low pg/mL levels whereas other antibody combination may require high pg/mL or possibly ng/mL levels for detection. Instead of normalizing data to the reference spots or experimental positive controls, background signal should be substracted from each analyte spot using the average pixel denisity from a clear arrea of the array.
The specific protease inhibitors are what were used during development and validation of the array. It is recommended to use the same or equivalent inhibitors, or consult the literature or empirically validate the use of other protease inhibitors or cocktails.
Lysis Buffer 6 contains phosphatase inhibitors but not protease inhibitors. If desired, protease inhibitors can be added to the lysis buffer immediately prior to use. We recommend 10 μg/ml Aprotinin (Tocris; Catalog # 4139), 10 μg/ml Leupeptin (Tocris; Catalog # 1167), and 10 μg/ml Peptstatin (Tocris; Catalog # 1190).
Array products simultaneously detects multi analytes in a single membrane. Every antibody pair used in Array has different affinity to their analyte. In addition, the abundance of every analytes may vary in samples. Therefore, one exposure time may not be optimal for all the analytes. R&D Systems recommends exposing membranes to X-ray film for 1-10 minutes with multiple exposures to get the best representation of low and high abundance analytes.
The general protocol provided in the Array kit booklet for tissue lysate preparation can be used for preparing adipose tissue. R&D Systems' lab has used the protocol for adipose tissue and did not remove fatty acids and triacylglycerols after homogenization.
R&D Systems Proteome Profiler Arrays are originally developed for chemiluminscent detection. However, many of the Arrays can be adapted to near-infrared fluorescence detection using the LI-COR Odyssey Infrared Imaging System. To achieve this adaption, the HRP-conjugated Streptavidin provided in the kit is replaced with IRDye 800CW Streptavidin, and the arrays are scanned using a LI-COR Odyssey IIS. Please refer to the protocol: https://www.rndsystems.com/resources/technical/use-proteome-profiler-arrays-li-cor-detection.
The software used to analyze Proteome Profiler arrays in-house is Quick Spots; manufactured by Western Vision. The Quick Spots product information may be found at http://www.wvision.com/QuickSpots.html.
These arrays are not a quantitative measure of protein expression, and it is difficult to determine a sensitivity for each of the analytes because the sensitivity is expected to vary depending on the analyte and the sample. Essentially, our arrays allow investigators to see a difference in relative expression levels of the analytes between a control/untreated/healthy sample when compared to an experimental/treated/diseased sample. R&D Systems have specifically optimized the antibody pairs individually to maximize the sensitivity while still minimizing the cross-reactivity with other analytes on the array. However, we do not test the sensitivity of each analyte. Ultimately, for best results, we recommend analyzing the membrane with different exposure times to determine which exposure time works best for your specific samples that you are testing.
The donut effect observed in the images is perfectly normal and is related to how the capture antibody dries as part of the printing process. Similar subtle ring effects may be observed in the representative data included in the insert. We recommend measuring the average pixel density across the entire spot instead of doing a point measurement.
It can be challenging to directly compare Western blot results to Arrays. Confounding factors include: the Array detects the protein in its native form while Western Blotting detects linearized and reduced protein; the matrix environment is not optimized for any one analyte; and the antibody pair may differ and have different sensitivity. R&D Systems recommends using a Duoset or DuoSet IC products for analytes of interest for users interested in confirming the Array results. In these ELISAs, the protein will be in a form similar to the one that is detected in the Array. Duoset ELISAs include a standard allowing the user to quantify the protein.