by Caitlin Smith
One of the gold standards for measuring protein levels in a biological sample is still the tried-and-true Western blot, also known as a protein immunoblot. In Western blotting, proteins are separated according to molecular weight by gel electrophoresis, then transferred from the gel to a blotting membrane (such as nitrocellulose or PVDF membranes). Proteins of interest are probed with specific antibodies, which can then be detected in myriad ways. One of the most common detection methods is chemiluminescence. In chemiluminescent detection, the primary antibody is generally detected with a secondary antibody that has been conjugated to an enzymatic reporter molecule; two common reporters are horseradish peroxide (HRP) and alkaline phosphatase (AP). When the enzyme is exposed to the appropriate substrate, a light-producing chemical reaction occurs. In other words, a chemiluminescent signal is generated. ECL™ reagents are some of the more popular chemiluminescent detection reagents. “Western blotting reagents as a whole have become quite specialized, with HRP substrates, washing and blocking buffers optimized for different imaging systems, sample types and sample abundances,” says Tamara Golden, marketing manager at Advansta. “This gives the scientist the ability to select reagents that best guarantee the success of any given experiment.” It also makes it hard for a Western blotting neophyte who must choose which detection reagents to use. The questions that follow may help you zero in on the best reagents for you.
HRP or AP: Which reporter enzyme should I use?
When choosing a reporter enzyme, you first need to consider approximately how much protein you expect to detect, and whether you will need to image the blot multiple times or over a period of days.
Generally, without special reagents, HRP blots can detect down to 1 to 3 pg, and AP can detect as little as 10 pg. “HRP is comparatively more sensitive and gives a signal of a shorter duration,” says Lawreen Asuncion, product manager at Bio-Rad Laboratories. Bio-Rad’s HRP-based Immun-Star™ WesternC chemiluminescence kit uses an enhancer solution to boost the signal and thus has even greater sensitivity, down to femtogram levels of protein detected.
“In contrast [to HRP], AP has a lower sensitivity but gives a longer and more stable signal, which can be useful if you need to re-image the blot days or even weeks later,” Asuncion adds.
Film or imager: Should my detection method influence my reagent decision?
There are two main ways to read chemiluminescence signals: film or digital imaging using a CCD camera. Although most chemiluminescent reagents can be used with either film or an imager, some reagents have been optimized for one or the other.
Film is the traditional Western blot readout, but the benefits of digital imaging are increasingly apparent. “Researchers view film as sensitive, but it is hard to find the optimal exposure, and you can only get a qualitative assessment of your blot,” says Asuncion. “It has been difficult to completely convert researchers over to CCD cameras— as most institutions still have a functional darkroom. While adoption of digital technology has been greatly accelerated by improved workflow, ease of use, and more flexibility in their product offerings, the cost of an imaging system is an obstacle that is preventing some researchers from being able to switch to digital imaging.”
Nonetheless, film imaging is preferred in some instances, such as when it is not possible to purchase equipment for CCD imaging. Using film is also a quick and easy way to query the presence or absence of a protein. Advansta’s “WesternBright ECL is designed for film-imaged blots,” says Golden. “It provides an extremely strong signal for greater sensitivity, allowing the researcher to load less protein, image with shorter exposure times or use up to 10 times less antibody than with others’ substrates. If the researcher plans to conduct a large number of routine blots and to image the blots with film, then an economical substrate such as WesternBright ECL may be preferred.”
If you plan to compare the levels of proteins between samples, you would be better off using a CCD imaging system, because it gives a broader linear dynamic range. “Quantitation is inaccurate on film, because of the limited dynamic range,” notes Asuncion. “CCD technology gives you a much broader dynamic range.” This is important for estimating the amounts of two or more proteins relative to each other on the same Western blot. “A broad, linear dynamic range allows you to compare weak and strong bands on the same blot and requires that high-end signals are not saturated and that the lower limit of detection is as low as possible,” says Frida Sandegren, product manager at GE Life Sciences.
The Amersham ECL Prime Kit was recently introduced by GE Healthcare Life Sciences for chemiluminescent detection. Both film and CCD camera can be used, but to properly quantify the results of your Western blot, a CCD camera is the recommended method for capturing the results. “It is at least twice as sensitive as the Amersham ECL Plus kit, with a limit of detection in the low picogram range,” says Sandegren. “Amersham ECL Prime is characterized by greatly increased signal stability that makes it possible to do repeated exposures and process several blots in one experiment. In addition, the three- to five-fold increase in intensity of the signals emitted by Amersham ECL Prime means that proteins can be detected using primary and secondary antibodies at three-fold higher dilutions than with Amersham ECL Plus. This reduces background and consumption of expensive antibody reagents.”
Advansta’s WesternBright Quantum chemiluminescence kit also is designed for CCD imaging. “It has a long-lasting, stable signal, so the scientist does not have to rush to image the blot but can image the blot several hours after substrate incubation, [which is] especially helpful when waiting for a shared imaging system to be free,” says Golden. Indeed, chemiluminescent technology is generally improving signal stability so that blots can be imaged for longer. “Chemiluminescence is based on an enzymatic reaction, and the intensity of the signal decreases over time as substrate is consumed,” notes Sandegren. “So the slower the turnover, the longer the signal remains at its most intense, meaning that bands corresponding to even the tiniest amounts of protein remain detectable for longer. And more stable signals mean you have more time to analyze your blot—less rush, fewer mistakes.”
Should I always choose the most sensitive reagents available?
Not necessarily. In general, the more sensitive your probes are, the harder it may be to keep the signal-to-noise ratio high enough to read the results above background with reliability. For example, if you expect to be seeing tens of picograms, and you want a longer-lasting signal, then maybe an AP-based kit is best for you. You can derive greater sensitivity from an AP blot by running the reaction for a longer time. However, the downside of this strategy is that it can lead to higher background signals, reducing your signal-to-noise ratio. Similarly, with the greater sensitivity of HRP reagents in general, you may run the risk of high background if you overexpose your film. These problems can be at least partially mitigated by paying attention to two mundane but important steps: washing and film exposure (if relevant).
Every Western blotting protocol includes washing steps eliminate unbound antibodies and reduce background signals. But the right amount of washing must be identified. Too little means a higher background signal; too much runs the risk of washing away some of your antibody or antigen from the blotting membrane, resulting in decreased sensitivity. There is no magic bullet. While standard washing conditions work well for many blots and are a good place to start, optimal conditions must be determined empirically for each blot.
To achieve the greatest sensitivity, the duration of film exposure also must be optimized. If the film is underexposed, the signal will not be strong enough to be read reliably. If the film is overexposed, background signals may be high enough to drown out the signal of interest. Also, protein bands that are close together may become so blurred as to be inseparable. In such a case, you might find help in a film treatment compound such as Thermo Scientific Pierce's Background Elimination reagent. This lets you optimize your exposure time after taking the film, by reducing the film exposure time while watching the reaction, and stopping the reaction when the ideal levels of signal and background for that particular blot are reached.
What are these special boosters and blockers?
Your signal intensity will vary according to individual conditions, but there are additional agents you can use to boost the signal you want to see and block out noise you don’t want to see.
To help with high background noise or low signals, EMD Millipore offers its Bløk and Signal Boost reagents, respectively. “Our Bløk Noise Canceling Reagents decrease the background seen on blots due to nonspecific antibody binding without leaving a thick, sticky layer like milk. Bløk is the only protein-free noise-canceling reagent available, which means you can stain after immunoblotting,” says Michele Hatler, product manager for Western blotting solutions at EMD Millipore. “Our Signal Boost™ Immunoreaction Enhancer is used during the primary and secondary antibody incubations steps and increases the binding efficiency of the antibodies to their target epitopes, hence increasing the signal on a Western blot. This allows you to use less antibody and/or to detect previously undetected bands without increasing your workflow.” EMD Millipore also offers its Luminata™ line of substrates with differing sensitivities, including the Classico, Crescendo and Forte.
A similar type of reagent, Thermo Scientific Pierce SuperSignal Western Blot Enhancer, “increases detection sensitivity while at the same time [decreasing] background in Western blots by improving specificity between a target and its antibody,” says Steve Shiflett, technical product manager for Thermo Scientific Pierce Products. “This product virtually eliminates problems associated with overexposure using X-ray film. It’s ideal for researchers who are using antibodies that bind nonspecifically.” If your target is low in abundance, you might benefit from SuperSignal Western Femto Substrate, which provides greater sensitivity. Additional Western blotting reagents that Thermo Fisher Scientific has introduced recently include Pierce ECL Plus Substrate, which is an acridan-based substrate that generates a signal when it reacts with HRP. “SuperSignal Chemiluminescent Molecular Weight Ladders help researchers determine the molecular weight of target proteins directly on the X-ray film,” says Shiflett. “We also now offer more than 10 different protein ladders for easy determination of molecular weight of target proteins in gel or on membrane.”
Future directions
Western blotting is becoming faster and increasingly more sensitive. Yet there are many ways in which it could be improved, and researchers should be on the lookout for new developments. One of the biggest challenges in chemiluminescence detection is being able to measure more than one protein at a time. “Looking at one protein is straightforward, but in order to study a second protein, the membrane will need to be stripped and then re-probed with different antibodies,” says Asuncion. “Not only is this time consuming, but certain membranes such as nitrocellulose typically do not hold up well under these conditions.”
In addition to detecting more than one protein at a time, Westerns could greatly benefit from the ability to detect more than just the level of a protein, according to Hatler. Such a development may demand the incorporation of different types of technologies. “This will require tools to detect multiple proteins within the same sample, modifications associated with those proteins, multiple post-translational modifications of the same protein, increased throughput of Western blots and the ability to maximize the amount of data from a single gel.” Until such lofty goals can be achieved, the considerations here will help you get started.
The image at the top of this article is Advansta's WesternBright ECL HRP Substrate.