Practical Tips for Nonisotopic Detection

Ambion's BrightStar® BioDetect™ Kit has been optimized to reduce the variables which have contributed to high background seen in nonisotopic detection systems of the past. We routinely use this system for both Northerns and nuclease protection assays to achieve sensitivities comparable to a 1 -2 day exposure using ³²P-labeled probes. Following the procedure as outlined in the BioDetect™ Instruction Manual and paying careful attention to the details outlined below, should result in a optimal signal-to-noise ratio for detecting even rare messages.

• Positively charged nylon membrane is the optimal membrane choice for use with Ambion's BioDetect Kit.
• Wear gloves when handling membrane. Avoid bending or crinkling the membrane. Handle the membrane by its edges with forceps - glove-prints can produce smudge background.
• Membranes should be free of dust and debris which may cause speckled background - rinse with 1X TBE if necessary.
• Handle the gel carefully post-transfer — gel fragments on the membrane can contribute to background. A quick rinse in high salt buffer post-transfer will help eliminate any gel fragments.

• Prior to nonisotopic detection complete all washes associated with the hybridization protocol (e.g. the low and high stringency washes performed after Northern blot hybridization). The detection washes do not substitute for any of the post-hybridization washes.

• Containers should be clean, free of lint and RNase-free. Treat with RNaseZap® and rinse well to remove residual conjugate before reuse.
• Containers should be slightly larger than the membrane to minimize solution volumes while keeping the membrane wet and free-floating.
• Avoid conical tubes as membranes may stick to the tube walls and not get sufficiently bathed in block and wash solutions.

• Dilute concentrated blocking and washing solutions in water - unsterile water can include alkaline phosphatase as a contaminate.
• Prewarm block and wash solutions to ensure components are completely dissolved - particulates can cause spotty background. Do not prewarm conjugate solution, it is an active enzyme and heating can damage it.
• Perform washes at room temperature.
• Monitor washes for the formation of precipitants. Perform incubations in draft-free areas away from A/C vents and warm wash and blocking solutions to 37°C if needed to dissolve precipitants. Do not warm conjugate, as this may reduce signal by inactivating the alkaline phosphatase enzyme. Modest evaporation of solutions during long incubations combined with air conditioning can frequently cause precipitant formation. At Ambion, we solve this problem by moving incubations to a warmer part of the lab (near a window) or by warming solutions for a few minutes during the incubation with a blow-type hair dryer.
• Dilute conjugate in a separate container and use a sufficient volume to keep the membrane wet and free-floating. Limit conjugate incubations to 30 minutes.
• If the conjugate is over 3 months old, protein aggregates may have formed which can contribute to speckled background. The conjugate can be centrifuged for 1 min to pellet any aggregates.
• Incubation with the substrate (CDP-Star®) can be done flat on plastic wrap. After incubation, drain excess substrate and blot briefly (2 sec.) between two sheets of filter paper. Do not overdry, the membrane must remain damp for the chemiluminescent reaction to proceed.

• A typical initial exposure time is 30-60 minutes. Emission peaks in 2-4 hours and lasts for several days. The best time to get high signal-to-noise ratio is during peak light emission. Exposure times will vary from 5 minutes to overnight.
• Expose film at room temperature, colder temperatures will slow enzyme activity or destroy it altogether resulting in decreased or absent signal.
• Intensifying screens used with isotopic probes will not improve exposure time or intensity of chemiluminescent reactions.