Historically, nonisotopic detection systems have been plagued by high background and low sensitivity. This poor signal-to-noise ratio has previously made nonisotopic systems an impractical choice for detecting small amounts of RNA or DNA. Ambion has now introduced the BrightStar® BioDetect™ Kit for the chemiluminescent detection of non-isotopic probes, which has been optimized to reduce many variables which lead to high background. Ambion also offers BrightStar®-Plus Positively Charged Nylon Membranes for which the detection kit has been optimized. The BioDetect Kit utilizes a streptavidin-alkaline phosphatase conjugate (SA•AP) which binds to biotin, and a light emitting substrate (CDP-Star™) activated by the alkaline phosphatase. This system affords sensitivity rivaling conventional 32P-labeling. The typical rule of thumb is that if a signal generated by a 32P-labeled probe can be detected in an exposure of 2-3 days or less, then it should be possible to perform the assay nonisotopically. However, as with any nonisotopic detection scheme, there are critical variables that affect sensitivity. With this in mind, we have compiled the following tips and hints, which we have found to minimize background and increase sensitivity.
Understanding the Chemiluminescent Reaction
- A nonisotopic label such as biotin is incorporated into or crosslinked (postsynthesis) to an RNA or DNA probe.
- This labeled probe is hybridized to the target RNA or DNA on a positively-charged nylon membrane.
- A conjugate of the enzyme, alkaline phosphatase (AP), and a ligand (e.g. Streptavidin), with high binding affinity for the label (biotin), is bound to the label on the probe.
- A substrate (CDP-Star) is added which alkaline phosphatase degrades, resulting in light emission.
- The membrane is exposed to film.
Principal Causes and Appearance of Background
- Non-specific binding of the biotinylated probe to the membrane during hybridization (seen in membrane hybridization assays, e.g. Northern blotting),
- Non-specific binding of the conjugate (SA•AP) to the membrane, and
- Non-enzymatic light generation by the chemiluminescent substrate (CDP-Star).
Background usually appears as:
- A generalized background across the membrane,
- Large smudges or splotches, or
- Smaller spots or speck on the membrane.
Before Beginning the Detection System
- We find that positively-charged nylon membranes consistently give the lowest background and highest signal-to-noise ratio when used with the BioDetect Kit. However, even among different manufacturers' positively-charged nylon membranes, we see variability in the signal-to-noise ratio. After testing the different membranes available, we identified the membrane with the highest, consistent signal-to-noise ratio, and we provide these membranes as part of our BrightStar System (BrightStar-Plus Membranes).
- Damage to the membrane can result in non-specific background in the form of spots and smears. Always wear gloves when handling membranes; it is preferable to manipulate the membrane at one corner with forceps.
- Membranes should be free of any dust or debris. If necessary, briefly rinse the membrane with 1X TBE before using.
- Avoid breaking the gel while disassembling the transfer setup. One way to do this is by immersing the whole stack (filter papers, gel and membrane) in 1X transfer buffer before separating the gel from the membrane.
- We find that small fragments of gel can stick to the membrane during the transfer step. Once cross-linked, this debris is not easily removed and leads to background which appears as small specks or spots on the membrane. Washing the membrane in 1X TBE for 3 5 minutes after the transfer, but before cross-linking, will help to remove fragments of gel which have adhered to the membrane and reduce background.
Tips for hybridization steps (for membrane hybridizations):
- Before starting the detection protocol, it is important to complete all steps and washes associated with the hybridization protocol. The BioDetect Kit does not substitute for any of the wash steps in a Northern or Southern blotting procedure. Do not confuse the blocking and washing steps of the hybridization protocol with those in the detection protocol, as they each serve a different purpose.
- Appropriate blocking of the membrane is necessary to prevent nonspecific binding of the probe to the membrane (i.e. use DNA blocking agents with DNA probes and RNA blocking agents with RNA probes).
- The correct hybridization and wash stringencies should be chosen to reduce nonspecific binding of the probe to nonhomologous sequences. See your hybridization protocol, or Ambion's Technical Bulletin #169.
- Use 10 ng of nonisotopic probe per 1 ml of hybridization solution for membrane hybridizations. Increasing probe concentration will not improve sensitivity, only background. If Ambion's ULTRAhyb® is used in membrane hybridizations, then the amount of nonisotopic probe should be scaled back to 0.1 ng/ml hybridization solution.
Suggestions about containers used for detection:
- Containers used for BrightStar BioDetect incubations should be clean and free of lint or other debris. Containers should be washed immediately prior to use, and may also be treated with a 0.4 M NaOH solution or Ambion's RNaseZap® to remove contaminating RNase. We recommend that containers be rinsed thoroughly again after use to remove residual conjugate.
- The containers used for BrightStar BioDetect incubations should be only slightly larger than the membrane being processed to minimize solution volumes, yet ensure that the membrane is completely wet and free-floating during all incubations. Higher volumes of BioDetect buffers may be used if necessary to accommodate larger containers.
- Do not perform the detection procedure in conical hybridization tubes as the membrane may stick to the walls of such tubes, preventing adequate washing of the membrane and resulting in higher background.
Preparing the detection solutions:
Since particulates can cause background spots, all buffer components of the BrightStar BioDetect Wash and Blocking Buffers should be completely in solution before beginning the detection protocol. This applies to both the concentrated (5X) and diluted (1X) Wash Buffers. If they are not, then incubate at 37-68°C until no precipitates are present. However, the temperature of the buffers should be 25-37°C when applied to the membrane. Note that the BioDetect reagents are already all supplied as solutions that have undergone fine filtration in order to eliminate solubility problems.
Ambion's BioDetect Kit has been optimized so that all washes, incubations and exposures to film should be carried out at room temperature (22-25°C). Incubators set to this temperature range can be used and may improve results, but are not necessary.
Precipitates during wash steps:
Since the washing and blocking steps involve using small volumes with large surface areas, evaporation can occur, leading to a decrease in the solution temperature and precipitation of solution components. If precipitation occurs, background may increase. Temperature controlled incubators are not usually necessary, but try to set up incubations in a draft-free area of your lab, avoiding running A/C vents. The washes should be performed at about 22-25°C. If the lab is significantly colder, then an incubator set to 25°C may help to reduce background. If precipitates are seen, briefly warm the solution and membrane to 37°C to resuspend the precipitate. Do not exceed 37°C, as this may reduce signal by inactivating the AP.
- Since the conjugate contains alkaline phosphatase, it should be treated and stored as an enzyme (i.e. keep on ice until used and store at 4°C).
- During prolonged storage periods, individual SA•AP conjugate molecules can aggregate, which can cause spotting or edge effects on the membrane in the downstream detection step. The tube of SA•AP should be spun prior to use in order to pellet any aggregates to the bottom of the tube, leaving individual free-floating SA•AP molecules that can be pipetted off the top of the liquid in the tube.
- The 1:10,000 dilution of the streptavidin•alkaline phosphatase conjugate should be prepared in a separate container before it is added to the membrane. Do not store dilute stocks of the conjugate.
- It is important to use a sufficient volume of Blocking Buffer for conjugate dilution such that the membrane remains wet and free-floating during the incubation.
- Limit the conjugate incubation to 30 minutes as indicated in the BioDetect Instruction Manual. While shorter incubation times may decrease background, sensitivity will also be sacrificed.
At the end of the incubation with CDP-Star, drain excess CDP-Star from the membrane by holding it over the incubation tray with forceps for 30 sec. Then briefly blot the membrane between two sheets of filter paper (1 2 sec) so that the final membrane appears damp but not wet and shiny.
- When exposing the membrane to film, it is important to remember that the specific and non-specific signal generation by the chemiluminescent substrate is faster than that generated by radioisotopes. Therefore, a typical exposure length for a radioactively labeled probe will be too long for a nonisotopic probe. An initial 30–60 minute exposure of the membrane immediately following incubation with the chemiluminescent substrate will give a very high signal-to-noise ratio; however, the sensitivity of detection will not be as great as that attained with exposures during peak light emission. Peak light emission is achieved 2–4 hours after the addition of the substrate and lasts for several days. During this time, short exposures of 1–30 minutes (which are comparable to overnight exposures with 32P-labeled probes) are typically sufficient for a highly sensitive exposure as well as a high signal-to-noise ratio. Longer exposures may be performed, but non-specific background will increase along with the signal.
- Since the cleavage of CDP-Star substrate by AP is an enzymatic reaction, the exposure of the membrane to film needs to occur near room temperature. If the exposing film is mistakenly placed below this temperature (e.g. -80°C, as is the case with radioisotopic probes), the AP will not be active and no light emission will occur. We find that exposing the membrane to film at temperatures > 25°C increases background.
- Note: Normal intensifying screens used with isotopic probes will not improve exposure time or intensity of chemiluminescent reactions.