Western blotting remains the most commonly used method for separating and detecting proteins, and yet it can be very difficult to make it work to your advantage. Optimizing your protocol is crucial, but, as stated by Ryan Short, imaging system marketing manager at Bio-Rad, “Most researchers aren’t Western blot technologists – they are trying to answer bigger questions.” Ryan goes on to say, “The problem is that most folks don’t know what needs to be optimized. And quite frankly, the Western blot process is too long. If each blot takes you two days, how much time can you really spend to optimize a Western blot?” Of course, factors such as antibody quality, blocker buffers, and transfer efficiency play key roles. Let’s take a closer look at the optimizing tips and tools that are available today.
Load the right amount of protein
Though optimization is crucial, the length of the traditional Western blot procedure makes it difficult. Short believes that one of the most important ingredients in a successful Western blot protocol is determining the protein load in each gel. “That’s critically important, because most people doing Western blotting have a protein of interest, and then they have to normalize their results to a housekeeping protein,” says Short. “If your protein of interest is a low-expressing protein, and your housekeeping protein is a high-expressing protein, you have to have both proteins be within the dynamic range of the assay, for the results to be valid. You probably need to load a lot of your low-expressing protein, maybe 50 - 60 µg protein, but at those loads, the housekeeping protein signal may be far outside the linear range of the assay. Understanding those dynamics are critical to having a good output for your Western blot.”
Efficient blocking: look beyond nonfat milk
Using the right blocking buffer can make a big difference. Though milk is often used, it is usually not as good as the high-quality commercial blocking buffers available today. “The blocking buffer should bind to regions on the membrane that could interfere with appropriate antibody binding,” says Priya Rangaraj, market segment manager for protein detection for protein biology products at Thermo Scientific. “If it does not, you will see speckling and increased background.” Blocking buffers for Westerns include the Thermo Scientific StartingBlock Blocking Buffer, the Odyssey Blocking Buffer from LI-COR Bioscience, and the PBS with 1% Casein blocking buffer from Bio-Rad. “Researchers need to take the time to include appropriate controls and test a variety of primary antibodies and blocking buffers,” agrees Shawn Mischnick, technical product specialist for reagents and accessories in biotechnology at LI-COR Biosciences. “Antibody performance can be significantly improved with the correct blocking buffer.”
Chemiluminescent detection: choose reagents with a prolonged, stable signal
Another important point to optimize, says Dmitry Bochariov, principal scientist at Advansta, is the quality and stability of the signal you measure. “Our substrates have longer and steadier signals, which help reduce variables associated with inconsistent times between the incubation with a substrate, and the moment when the blot can actually be imaged or exposed to the film,” says Bochariov. “These variables are very difficult to control and reproduce because they involve manual handling of blots, covering with protective wrap, affixing to the cassette support or setting up an imaging system, and other human factors. Long and steady chemiluminescent light output can greatly compensate for all these factors.” Another example is the Thermo Scientific SuperSignal Substrates for HRP, available in three varieties for different target levels (picogram, mid-femto gram, and low femtogram).
Another tool for boosting signals of low expressing proteins is the new Thermo Scientific SuperSignal Western Blot Enhancer. “The enhancer not only increases signal by making epitopes more available to the primary antibody, but also reduces background,” says Rangaraj. “It is particularly useful with low abundance, or weakly immunoreactive, antigens or proteins.” Also new is their Thermo Scientific MYECL™ Imager, which digitally images, detects, and archives Western blots. “The CCD-based imaging system offers twice the sensitivity of traditional x-ray film, with 10 times the dynamic range,” says Rangaraj.
Consider fluorescent detection
Many researchers are not taking advantage of the full range of labels available, according to Stephanie Noles, global in vitro product manager at Carestream Molecular Imaging. “Transitioning over from chemiluminescent labels to fluorescent labels can be daunting, but it really opens up the type and amount of research that you will be able to accomplish,” she says. “More and more fluorescent antibodies are being made available for protein detection. New dyes, antibodies, and other labels are becoming available at such a rapid rate, you really want to be able to use anything that is out there.” Carestream Molecular Imaging’s new Gel Logic 6000 PRO imager is designed to help researchers strive for greater quantification, as they they become more interested in using fluorescent (rather than chemiluminescent) labels. “The Gel Logic 6000 PRO has the ability to detect all fluorescent labels as well as chemiluminescent labels on the same blot,” she says. “This greatly expands the research scope, as it will prevent the need for the stripping and reprobing of blots that is commonly required with HRP-type antibodies.” The imager also lets you overlay fluorescent and luminescent signals without ever having to move your blot, or strip and re-probe, so you can detect more proteins simultaneously.
Streamline your protocol
Reducing the amount of time that Western blots take from start to finish makes optimization much less painful. Bio-Rad’s V3 Western Workflow – three Vs for visualize, verify, and validate – accomplishes this in two ways. Their stain-free technology uses a UV-activatable compound embedded into Bio-Rad’s pre-cast gels that allows you to visualize the proteins throughout the Western blot experiment, giving you a means to check the progress of the blot at different points in the process. This means that rather than run two gels and stain one with Coomassie Blue in order to check the other one, you can run one gel and simply put it into an imager to activate with UV light for one minute. You can also image the blotting membrane and the gel after transfer to assess transfer efficiency.
“We are adding checkpoints to the process, but not adding additional time to the process,” says Short. “In fact we’re actually speeding it up.” Traditionally, polyacrylamide gels take about 40-60 minutes, but Bio-Rad’s gels now take 15 minutes or less. Their TransBlot Turbo Transfer System also reduces transfer time from an hour to about 3-7 minutes. “With shorter protocol times, it is easier to optimize,” he says. “People are scared to do more optimization, because they think it’s going to add more time to the blot process. With our system, if it’s not going well, it’s easy to do it again. Not only do you have more time to optimize, you also have more checkpoints to evaluate which step in the process needs optimization.”
Another challenge in optimizing Westerns is normalizing low-expressing proteins using an abundant housekeeping protein within the assay’s dynamic range. “With our stain-free technology, instead of normalizing to housekeeping protein, we use total protein normalization,” says Short. “This is especially helpful for people doing chemiluminescence detection, where people are stripping the antibodies for the protein of interest off the blot after detection, and then re-probing for their housekeeping protein, which takes 2-4 hours. With stain-free technology, you don’t have to go back and do the strip-and-reprobe step.” Instead, you image your protein of interest and the total protein on the same blot simultaneously, and then normalize.
Bio-Rad’s newest tool in this process is software called ImageLab 4.1, which automates the process of blot normalization in a more intuitive way than other software tools. “Instead of manually drawing boxes around housekeeping proteins, Image Lab boxes each lane and calculates total protein load.” says Short. “Algorithms then detect protein of interest bands on the blot and automatically calculate the normalized intensity values.”
Aldrin Gomes, assistant professor in the department of neurobiology, physiology, and behavior at the University of California Davis, who uses Bio-Rad’s V3 workflow in his lab, says it’s helpful for more than saving time. “We can also do more with the blotting system,” he says. “Before, we could only do 2 blots at time. With the Turbo Blot, we can do 4 mini-gels at one time (or one big and two small). This allows us to try different conditions so we can optimize faster. For example, we can adjust transfer times (5 min vs 7 min) or try different percentage gels.”
Mischnick agrees that one of the biggest limitations in blot optimization is time. “It can take up to 8 hours to generate and detect a Western blot,” she says. “LI-COR’s Quick Western Kit-IRDye 680RD provides a universal antibody detection reagent that can be combined with the primary antibody incubation step, eliminating the need for a secondary antibody, and saving up to 90 minutes on the total detection time.” In addition, the kit can detect primary antibodies from various hosts, and eliminates the need to purchase multiple IRDye secondary antibodies.
Multiplexing and automated solutions
The ability to run many samples simultaneously can help to speed optimization by allowing you to run samples in different conditions or different protein concentrations. The LI-COR MPX (Multiplex) Blotting System screens multiple samples and targets on a single Western blot. “The independent channels and multi-sample features of the MPX eliminate common limitations encountered with antibody cross-reactivity during standard Western blot analysis,” says Mischnick. “The MPX protocol offers customers the ability to optimize their Western blot conditions in one experiment and works with all detection formats.”
For high-throughput optimization, ProteinSimple’s Sally is a completely automated Western blotting instrument. Your role is to load protein samples into a 384-well plate, then put the plate into the instrument. The rest is done by Sally. “It draws your proteins into the capillaries, which are the equivalent of lanes, where we do the size-based separations,” says John Proctor, director of corporate development at ProteinSimple. “It does the immunoprobing and the secondary antibody, and it takes all the pictures. The user just comes back to a series of images - it will show you long and short exposures of all the capillaries you've run.”
The Sally system sports other advantages too. Traditional Western blots use one primary antibody at a time, but Sally can use multiple primary antibodies per capillary, or across different capillaries. “For example, the Sally system can run 96 capillaries simultaneously, which means you could run 96 primary antibodies against the same sample in one experiment,” says Proctor. “It gives you a lot of flexibility to use different primary antibodies, different buffer types, different sample types, different secondary antibodies. Because you have a lot of capillaries to work with, you can make quadrants, or a matrix, or different conditions, and then in one experiment arrive at a final fully optimized assay.” What Sally may lack in speed, she makes up for in throughput (96 capillaries takes 18-19 hours, or an overnight run). ProteinSimple’s lower throughput instrument, Simon, runs 12 capillaries in 3-5 hours. “Sally has the equivalent overall time of a traditional Western, but now we’re focusing on how to make the process faster for people, maybe 5-6 hours instead of overnight,” says Proctor.
Whether choosing a traditional or high-throughput Western blotting system, one of the most important factors in good optimization is to know what you are trying to do, and why. “It is important to really understand fundamental principles behind all techniques and steps involved,” says Bochariov. “If a researcher does not understand well the principles and mechanisms of the natural phenomena that make the assay work, they will have difficulty distinguishing a really wonderful effect that may lead to a discovery, from just a plain experimental artifact.”
The image at the top of the page is from Bio-Rad's Immun-Star™ HRP Chemiluminescence Kits.