by Caitlin Smith
For decades, Western blotting (also known as protein blotting) has been instrumental in our ability to detect proteins separated by molecular weight from a protein mixture via SDS-PAGE. The ensuing immunoblotting step is still today’s foremost protein identification technique. (A close rival is mass spectrometry, but its high cost and requirement for specially trained operators precludes it from widespread use.) After a protein transfer step from the gel matrix to a membrane—which is usually made from either nitrocellulose or polyvinylidene difluoride (PVDF)— the protein blot is probed with antibodies labeled for subsequent detection. Today’s blots have evolved a great deal since their inception. The typical Western blot performed today is faster, more efficient and more sensitive than ever.
Choosing an apparatus based on speed and transfer efficiency
The two most important points to consider when choosing a Western transfer system are speed and transfer efficiency. Historically, Western blots have been divided into three categories based on the type of equipment and protocol they use. Generally, you can run Western blots using dry, semi-dry or wet (also called tank) systems. Each choice has advantages and disadvantages. Dry systems give the fastest transfers, but the lowest transfer efficiency. Wet transfer systems, in which transfer occurs with the blotting “sandwich” assembly submerged in a tank of buffer, give the best transfer efficiency (especially for a wide range of molecular weights); but wet transfer systems also use the longest and most laborious protocols. In the middle are the semi-dry systems, in which the gel and membrane are not submerged in buffer but are sandwiched between filter paper saturated with buffer. Semi-dry systems fall between wet and dry transfer systems in speed and transfer efficiency but are generally not as good for high molecular weight proteins. However, the limits of these transfer methods are being nudged by new technology that is making all of them faster and more efficient.
Priya Rangaraj, protein detection market segment manager at Thermo Fisher Scientific, says the choice of a dry, semi-dry or wet transfer system is often a personal one. “Researchers learn to do their transfers in a certain way, and they tend to continue to do them using the technique that’s most familiar to them,” he explains. “That said, our researchers typically choose a wet transfer if they’re transferring using a gradient gel or if they have really high or really low molecular weight proteins they’re trying to capture. Some researchers prefer wet transfer because they like the convenience of setting it up before leaving the lab overnight. Many researchers prefer the semi-dry transfer because it’s cheaper and uses less reagents. For everyday blotting, the fast semi-dry transfer systems provide convenience, speed and economy.”
Selecting the membrane and other accessories
Western blot accessories that you likely will need, and which definitely will make life easier, include blotting membranes and protein detection systems. Most Western transfer systems can use either type of blotting membrane (nitrocellulose or PVDF), so you need to choose which is best for your experiments. Comparing the two, nitrocellulose membrane is cheaper but more fragile—it cannot withstand repeated probings for immunodetection, for example. On the other hand, PVDF is sturdier and has greater protein binding capacity but is more expensive. One type of PVDF is Millipore’s Immobilon™ PVDF transfer membrane, which comes in three types. The Immobilon-P membrane (0.45 µm pore size) is suitable for a variety of blotting applications; the Immobilon-PSQ membrane (0.2 µm pore size) has a higher binding capacity and retention than the -P model, and is good for protein sequencing and blotting of low molecular weight proteins. The Immobilon-FL membrane (0.45 µm pore size) is designed for fluorescence-based blotting and has low background fluorescence across a wide range of wavelengths.
Additional useful adjuncts to protein blots are protein detection kits to make the detection step easier and faster. For example, EMD Millipore’s SNAP i.d. System uses the force of a vacuum to drive reagents through the blotting membrane. This makes the blocking, washing and probing steps faster than conventional blotting, which mostly uses diffusion to move the reagents. The SNAP i.d. System also more thoroughly washes and blots the membrane, because solutions are actively drawn through the membrane instead of just rinsing the surfaces. This reduces the total time for blocking, washing and antibody incubation to 30 minutes. Life Technologies also recently released its new iBlot® Western Detection Kit. “The new iBlot Western Detection kit speeds up the conventional immunodetection method from 3-plus hours to less than 30 minutes with comparable sensitivity,” says Samantha Li, market development manager for Life Technologies. An added bonus to these new protein detection systems is that it takes less time to optimize Western blotting conditions when setting up new experiments.
Dry transfer systems
The latest offerings in dry blotting systems include Life Technologies’ new iBlot® Dry Blotting System. Completely bufferless, the iBlot® transfers proteins from gels to membranes in seven minutes or less. When adding the detection step time to the blotting step, Li says that “the iBlot Dry Blotting System allows researchers to quickly perform immunodetection of transferred protein on nitrocellulose or PVDF membrane in less than 40 minutes.”
Li notes the importance of the quality of the transfer step. “The main use for Western blotted membrane is for immunodetection,” she says. “Therefore, ‘quality of transfer’ is looking at the sensitivity of immunodetection of the blotted proteins (or antigens). Quality of transfer is not the quantity of protein extracted from the gels, i.e., leaving ‘clean’ gels, but how well the proteins (antigens) are absorbed by the membrane, and [how well they are] exposed and made accessible for highest detection.” Usually, wet transfer systems offer high efficiency but at a cost of time and effort. Some researchers switch to semi-dry blotting but may pay for the time savings with a decrease in transfer quality. “Although both of these methods are commonly used, neither of them offers the high quality of transfer combined with speed and convenience of the iBlot Dry Blotting System from Life Technologies,” says Li.
Semi-dry transfer systems: A blot in 3 minutes?!
As different systems evolve, features from different methods are integrated. Bio-Rad’s newest addition, for example, resembles a semi-dry system but is much faster and offers better transfer efficiency than traditional semi-dry, particularly for high molecular weight proteins. In April, Bio-Rad launches its Trans-Blot Turbo Transfer System, which is easier, faster and delivers better transfer efficiency than many other transfer methods. The Trans-Blot Turbo Transfer System reduces the blot run time to as little as 3 minutes. “A blot in as little as 3 minutes?” you may ask. “How is this possible?” Jeff Xu, product manager for Western blotting at Bio-Rad Laboratories, explains how the company reduced the run time so dramatically. “It’s a combination of the new hardware and new consumables,” he says. “We developed a new formulation for the blotting buffer, and used a specialized material for the filter paper. That combination allows much higher amperage to be driven through the blot, resulting in not only faster but more complete transfer.” The system is different from any that Bio-Rad has offered before and includes an integrated power supply to satisfy the higher amperage requirement. “It includes two cassettes, and each cassette can accommodate one to two mini-gels, or one midi-gel,” says Xu. “These cassettes are independently controlled. Therefore, you can start transferring one gel while you set up the second cassette, and by the time you’ve finished the second cassette. Trans-Blot Turbo can also transfer up to 4 mini gels or 2 midi gels at the same time, doubling the throughput of most systems on the market.”
The benefits of Bio-Rad’s new system are echoed by a researcher who has sampled the model. “We use the Trans-Blot Turbo Transfer System in the place of traditional Western blotting systems,” says Ian MacRae, assistant professor at the Scripps Research Institute. “[It has] three main strengths: speed, convenience and reproducibility between users. The Trans-Blot Turbo System eliminates any user-to-user variation concerns, making it possible to create high-quality reproducible blots in a fraction of the time. [It] has allowed our researchers to streamline transfers, decreasing processing times from hours to a matter of minutes.” Another feature that makes Bio-Rad’s new system faster and easier is the use of prepackaged “Turbo Packs.” Made of specialized blotting paper, methanol-free buffer and membrane, the pre-assembled Transfer Packs save time in preparing the blotting sandwich by eliminating additional reagent and membrane preparation time. At the end of a run, you discard the used materials and open another Turbo Pack and begin a new transfer session right away. “Best of all, unlike other rapid blotters, the Transfer Packs generate minimal waste and are not hazardous to the environment,” says Xu.
Thermo Fisher Scientific also released a new semi-dry system recently. The Thermo Scientific Pierce Fast Blotting System transfers proteins in as little as 10 minutes. “The system includes the Pierce Semi-Dry Blotter and a methanol-free transfer buffer, which is really attractive to researchers because it’s more economical, and it doesn’t have the hassles associated with hazardous waste disposal,” says Rangaraj. “Our blotter enables researchers to transfer up to four mini-gels simultaneously and doesn’t require instrument-specific consumables.” Some researchers prefer not needing to purchase consumables to use a blotting system; others like the convenience and time savings. “When we were developing our Fast Transfer System, we asked researchers [what was important to them in choosing a transfer system,] and they identified price, speed, transfer efficiency and no requirements for instrument-specific consumables as key features to them,” says Steve Shiflett, M.S., protein detection technical product manager at Thermo Scientific.
Wet transfer systems
Though highly efficient, the longer run times of wet transfer systems usually lead researchers to set it up to run overnight or to run during the day while they do other work. Many manufacturers offer easy-to-use wet or tank transfer systems. For example, Life Technology’s XCell line has a new addition called the XCell SureLock® Mini-Cell. It uses a gel tension wedge to keep the polyacrylamide gel in place and to help seal the blotting assembly into its position in the buffer tank. Users of another model, the XCell II™, can purchase the SureLock Retrofit Kit to convert their system into an XCell SureLock. The Retrofit Kit contains a SureLock mini-cell lid, gel tension wedge and molded mini-gel buffer dam. “Recent innovations in precast gel technology have also accelerated the wet transfer process,” says Xu. “Bio-Rad Mini-PROTEAN TGX precast gels utilize a unique chemistry that allow tank transfer to be completed in as little as 15 minutes.”
Improvements in Western blotting systems are likely to keep coming. “Any time you can improve on speed and transfer efficiency,” says Shiflett, “researchers are going to be happier.”
The image at the top of this article is Bio-Rad's TransBlot Turbo Transfer System.