Lysis buffers packed in the Array Kits are lot matched components and are specific for the lot of Array kits in which they are supplied. For optimal performance of the Array kits, do not interchange or substitute Lysis buffers with those from other lots or sources.
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.
A number of the Proteome Profiler Array kits are validated for use with tissue lysates. Please review each kit's manual for validated sample types. If not listed as validated, the kit's compatibility with tissue lysates will need to be determined by the researcher.
Typically, any instrument used to analyze a Western Blot or which can measure chemiluminescence should be compatible with Bio-Techne's array kits. It is possible to image array membranes using a Chemi Doc system as several customers have reported success using this analysis system. Bio-Techne does not have specific recommendations in regards to settings to use, so analysis conditions will need to be optimized by the end user.
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 combinations 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 subtracted from each analyte spot using the average pixel density from a clear area 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.
The pixel densities of the Arrays' reference spots 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.
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 kits are designed to detect multiple analytes on a single membrane simultaneously, using different capture and detection antibodies. Each antibody pair has different binding affinity for the target analyte. Additionally, analyte abundance may vary in samples due to multiple factors. 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 representative signal for low and high abundance analytes.
R&D Systems defines array memebrane overexposure as when the two positive control reference spots merge and appear as a single large spot. Typically, this can be seen when membranes exposure time is > 10 minutes, but overexposure can also be seen earlier depending on other variables.
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.
Clear plastic sheet protectors can be purchased from office supply stores (e.g., Office Depot, Staples) or found on Amazon. For example, Office Depot Item # 491694 or the Ktrio brand of clear sheet protectors on Amazon are suitable. Clear sheet protectors can be trimmed down to the appropriate size. Please see the Array demo video found here (https://www.rndsystems.com/resources/protocols/profiling-changes-receptor-tyrosine-kinase-phosphorylation-using-antibody-arrays) and fast forward to approximately 6 min 20 sec mark. An example of a plastic sheet protector is shown.
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.
Bio-Techne uses Kodak x-ray film (Kodak® BioMax™ Light-1, Catalog # 1788207) or an equivalent. There is no particular brand that is recommended for autoradiography cassettes. What is important is to match the cassette size with the film size. For instance, some cassettes are small and fit only 5 x 7 inch film. Bio-Techne strongly suggests using cassettes that can accommodate 8 x 10 inch film for the flexibility that size provides.
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.
It is difficult to define sensitivity for each analyte in an array kit because Proteome Profiler Array is not a quantitative measure of protein expression. Furthermore, sensitivity for a given analyte will differ widely depending on many variables, including sample type, treatment conditions, sample storage and handling, and detection equipment (e.g., film vs digital cameras vs LI-COR, etc.). Arrays measure a difference in relative expression levels of analytes between a control/untreated/healthy sample when compared to an experimental/treated/diseased sample. The individual antibody pairs are optimized to maximize their respective sensitivity while minimizing the cross-reactivity with other analytes on the array.
At a minimum, all array kits have been validated with lysates from ligand-treated cells. In addition, some kits have also been validated with other sample types such as tissue lysates, saliva, milk, urine, serum, plasma, and cell culture supernates. Please consult kit specific product datasheet in the "Sample Collection & Storage" section for details on validated sample types.
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.