R&D Systems’ Proteome Profiler arrays are rapid, sensitive, and economical multiplex arrays for the simultaneous detection of many proteins in a single sample. Originally developed for chemiluminescent detection, many Proteome Profiler arrays are amenable to near-infrared (NIR) fluorescence detection using the LI-COR Odyssey® Infrared Imaging System. To achieve this adaptation, the HRP-conjugated Streptavidin provided in the kit is simply replaced with IRDye® 800CW Streptavidin, and the arrays are scanned using a LI-COR Odyssey Infrared Imaging System. (This protocol is not applicable to Proteome Profiler arrays that use an HRP-conjugated detection antibody instead of Streptavidin-HRP.)
An advantage of NIR detection is that protein expression can be measured over a much wider linear dynamic range compared to that allowed by chemiluminescence methods. This approach facilitates accurate analysis of weak and strong spots on the same membrane without the uncertainty and inconvenience of multiple exposures. In addition, NIR does not require film, chemicals for development, or the use of a dark room.
- Collect and store samples based on the directions in the product insert (kit booklet).
- Refer to the kit booklet for reagents provided and other reagents needed.
- Bring all reagents to room temperature before use and prepare reagents according to the directions in the kit booklet.
- Pipette array buffer into the dish as directed in the kit booklet.
- Using flat-tip tweezers, remove each membrane to be used from between the protective sheets. IMPORTANT: CAREFULLY CUT OFF THE STAMPED IDENTIFICATION NUMBER ON THE MEMBRANE USING SCISSORS. The dye used for the numbers on the membranes will fluoresce and interfere with LI-COR detection. If desired, the membranes may instead be labeled with pencil in the empty space to the right side of the array. Pick up the membrane from this edge during all processing steps.
- Continue to follow the directions in the kit booklet, completing the incubation(s) and steps to wash the membranes, and stop when reaching the steps to prepare and use Streptavidin-HRP. Streptavidin-HRP will not be used in this protocol.
- Dilute the IRDye 800CW Streptavidin (LI-COR, Catalog #926-32230) 1:2000 using the array buffer specified in the kit booklet for Streptavidin-HRP dilution.
- Refer to the volume of diluted Streptavidin-HRP the kit booklet specifies to be added to the 4-well dish. Instead, pipette this volume of diluted IRDye 800CW Streptavidin into each well of the dish.
- Carefully remove each membrane from its wash container. Allow excess buffer to drain from the membrane by blotting the lower edge onto absorbent paper. Return the membrane to the 4-well dish containing the diluted IRDye 800CW Streptavidin. Cover the wells with the lid.
- Incubate the membranes for 30 minutes at room temperature on a rocking platform.
- Wash each array as described previously in the kit booklet.
- Carefully remove each membrane from the wash container. Allow excess wash buffer to drain from the membrane by blotting the lower edge onto absorbent paper. Collect images with an Odyssey Imager.
The positive signals seen on developed film can be quickly identified by placing the transparency overlay on the array image and aligning it with the three pairs of positive control spots in the corners of each array. The location of controls and capture antibodies is listed in the Appendix of each kit booklet.
Suggested starting Odyssey scan parameters:
Resolution: 84 µm
Focus offset: 0.0 mm
Intensity: 5, adjust as necessary.
Collected images may be manipulated using Adjust Image Curve and Alter Image Display Odyssey settings.
Technical Hints & Limitations
- For research use only. Not for use in diagnostic procedures.
- This kit should not be used beyond the expiration date on the kit label.
- Do not mix or substitute reagents with those from other lots or sources.
- Any variation in buffers, operator, pipetting technique, washing technique, and incubation time or temperature can alter the performance of the kit.
- The membranes are validated for single use only.
- Always use gloved hands and flat-tipped tweezers to handle the membranes.
- A thorough and consistent wash technique is essential for proper assay performance.
- Wash buffer should be removed completely from the membrane before proceeding to the next step.
- Avoid microbial contamination of reagents and buffers.
- Soluble receptors and other proteins present in biological samples do not necessarily interfere with the measurement of proteins in samples. However, until these proteins have been tested with the Proteome Profiler Array, the possibility of interference cannot be excluded.
- Stamped identification number on membranes must be removed carefully with scissors prior to block step.
- Membrane may be labeled with pencil in the empty space to the right side of the array. Pick up the membrane from this edge during all processing steps.
- Do not allow the membranes to dry out during any step of the protocol. Membranes should be scanned wet and bubbles should be carefully removed when placing membranes on the Odyssey scanning bed.