|Date: March 15, 1999||Western Detection Kit Table|
After E.M. Southern devised a method to transfer DNA to a membrane, a technique that became known as Southern blotting, subsequently devised methods of transferring different moieties to membranes were wittily labeled after the remaining compass points. To "head West" is to separate protein samples by polyacrylamide gel electrophoresis and transfer them onto nitrocellulose, polyvinylidene difluoride (PVDF), or nylon membranes. Transfers can be carried out by diffusion, capillary action, or electrotransfer, although electrotransfer is the most efficient and widely used method. When the transfer is complete, the membrane can be incubated with an antigen-specific primary antibody (monoclonal or polyclonal) to form an antigen-antibody complex. The primary antibody is then recognized by a species-specific secondary antibody conjugated to a marker complex such as an enzyme or a radioactive isotope. The antigen-antibody-antibody complex can be detected directly, in the case of radioactive labels, or by colorimetric or chemiluminescent detection after the enzyme-catalyzed reaction of an added substrate. For Western blotting of biotinylated proteins, the primary antibody can be replaced by enzyme-conjugated streptavidin.
Additionally, proteins can be electrotransferred to membranes for subsequent amino acid or peptide analysis via MS (MALDI-TOF) and Edman degradation. With the recent push toward proteome analysis, requiring Western blotting of complex 2-D gels, optimizing Western blots becomes increasingly important.
The technique that is commonly referred to by EC Apparatus's trademarked name "electroblotting" is available in two methods for proteins: tank blotting and semidry blotting. Semidry blotting involves the use of a minimum amount of buffer and an easy-to-assemble blotting apparatus. Tank blotting, as the name suggests, is carried out in a tank of transfer buffer, and while it is more time consuming to set up, it offers more options, such as temperature, time, and voltage control. The following factors should be considered when determining which system to use:
- Buffer volume. The small amount of buffer required for transfer using a semidry system cuts the costs of reagents and saves time required to prepare large quantities of buffer. If your lab stock of buffer always seems to need replenishing when it's your turn to transfer a gel, you'll appreciate this feature!
- Electrodes. Plate electrodes, standard in semidry blotting systems, create a high-strength electrical field with higher current densities than wire electrodes commonly used in tank transfer systems. In semidry blotting, the gel and membrane are sandwiched between filter papers soaked in buffer, which serve as ion reservoirs. Because the plate electrodes are in direct contact with the filter papers, the field strength across the gel is maximized for fast, efficient transfers.
- Time. The small buffer capacity of semidry blotting systems limits the time over which the transfer can be carried out because longer blots deplete the available buffer. In the tank system, buffer is not depleted and transfers can be extended for up to 24 hours, allowing overnight use. However, if time is of the essence, semidry blotting provides rapid transfer--in as little as 15 to 30 minutes for minigels and 30 minutes to one hour for large gels. Tank transfers can take from 30 minutes to five hours, depending on the type of electrode used, gel thickness, electrical current distribution (mA/cm2), and the type of power supply used.
- Temperature. For transfers at low temperatures, (for example, for blotting native enzyme), buffer cooling is required, a feature not available in semidry systems. In tank systems, cooling coils and refrigerated water circulators are available. Cooling capacity is also an attractive feature if transfers are to be performed rapidly or if high molecular weight proteins are to be transferred (i.e., using high voltage).
- Protein binding. Using tank transfers, conditions can be adjusted so that low molecular weight proteins bind efficiently to the membrane. Using semidry systems, low molecular weight proteins may pass through the membrane. Because voltages are limited in semidry blotting by the lack of a cooling system, it may not be possible to transfer high molecular weight proteins using this system.
- Multiple transfers. Multiple transfers can be carried out using both the tank and semidry systems, but in the semidry systems, dialysis membrane is required for separation of gel sandwiches. Semidry blotting is a quick and convenient method of transfer, but for a more sophisticated approach, tank transfer remains the preferred choice. When transferring multiple gels in a stack using semidry units, the uniformity of the transfer varies from gel to gel; typically the gels closer to the negative electrode transfer less efficiently.
Bio-Rad offers three models of transfer apparatus: the Trans-Blot Cell, the Mini-Trans-Blot Cell, and the Trans-Blot SD SemiDry Transfer Cell. The Trans-Blot Cell, a tank system, offers several unique features to ensure maximum control over transfers. It comes with interchangeable plate or wire electrodes. Use of the plate electrodes--platinum-coated titanium for the anode and stainless steel for the cathode--results in faster, more efficient transfer of high molecular weight molecules. Both wire and plate electrodes can be moved closer together to increase transfer efficiencies in high-intensity applications. Power settings vary from 30 V (for reproducible, quantitative binding achieved by overnight use) to 200 V (for rapid transfers). The Super Cooling Coil and a water recirculator ensure thermal control for low-temperature and high-voltage transfers and prevent buffer depletion. For ease of assembly, a hinged gel-cassette clamping system is provided, which eliminates slipping and ensures proper contact between the membrane and the gel. Three gels, each up to 16 x 20 cm, can be blotted simultaneously in a tank volume of 4 L.
The Mini Trans-Blot Cell is designed for rapid high-quality blotting of one or two 7.5 x 10 cm minigels in a tank volume of 450 mL. The Bio-Ice cooling system absorbs heat generated during transfers. This system is a component of Bio-Rad's modular Mini-Vertical Electrophoresis System and is available either as a complete apparatus or as a module that adapts the Mini-PROTEAN II Cell for transfer applications, a useful option if you don't need a dedicated blotting apparatus.
The Trans-Blot Semi-Dry Transfer Cell is designed for fast, efficient, and economical blotting. Using plate electrodes similar to those used in the Trans-Blot Cell, transfers of gels can be carried out in 200 mL of buffer and completed in as little as 15 to 30 minutes for minigels and 30 to 60 minutes for full-size gels up to 20 x 18.5 cm. For high throughput applications, the platform can accommodate up to six minigels when arranged side-by-side. The use of Sequiblot PVDF membranes ensures nearly complete binding of all proteins. Bio-Rad has overcome the problem of short circuits across the electrodes by designing a single-step locking system that ensures even pressure across the blotting sandwich and uniform contact across the entire electrode surface.
Comparison of Three Chemiluminescent Substrates Three identical blots with serial dilutions of mouse IL-2 were incubated with SuperBlock Blocking Buffer, 100 pg/ml dilution of rat anti-mouse IL-2, 2pg/ml HRP-labeled goat anti-rat, followed by the three substrates indicated on the figure. Figure provided by Pierce Chemical Company.
Hoefer miniVE vertical electrophoresis system. Figure provided by Amersham Pharmacia Biotech
For some of the advantages of tank transfer, such as cooling capacity and buffer supply, coupled with speed approaching that of semidry transfers, the Mighty Small Transphor Tank Transfer Unit offers a valuable alternative. Up to four minigels can be transferred in as little as 30 to 60 minutes in only 1 L of buffer. An alumina-covered heat exchanger is built into the base. Tap water runs through the cooling channel and a magnetic stirrer is used for buffer circulation, ensuring no more than a 5°C temperature rise during typical transfers.
Two models of the SemiPhor Semi-Dry Transfer Unit are available: one for transfer of a 14 x 16 cm gel or two minigels, and one for gels up to 21 x 26 cm or four minigels. Both models are designed for fast, uniform transfer in less than an hour and include durable platinum and stainless steel electrodes. The uniform transfer is due to specially designed electrodes that minimize buildup of air bubbles during transfer.
As Bio-Rad's Mini Trans Blot Cell can be used to convert a vertical electrophoresis system into a tank transfer system, so Amersham Pharmacia Biotech's Hoefer miniVE vertical electrophoresis system can be converted into a tank transfer system by addition of Blot Modules. Each "semi-wet" Blot Module can transfer two gels using only 300 ml of buffer in as little as 45 minutes. Using two Blot Modules in the minVE, four gels can be transferred simultaneously.
Amersham Pharmacia Biotech's NovaBlot Kit can be used to convert the horizontal Multiphor™ II Flatbed Electrophoresis System into a semidry blotting configuration employing graphite electrodes. The Multiphor II Flatbed Electrophoresis System itself is extremely versatile and can be used for a wide range of applications, including 2-D electrophoresis, immunoelectrophoresis, SDS and native polyacrylamide gel electrophoresis using precast gels, and DNA electrophoresis.
Owl Separation Systems offers two tank transfer models: the Mini-Tank™ Electroblotter and the Bandit Large Format Electroblotter. The MiniTank can be used to transfer up to four minigels at once in 1,300 mL of buffer. The Bandit can be used to transfer one or two gels up to 20 x 18.5 cm in 3 L of buffer. Both the MiniTank and the Bandit have cooling chambers: the MiniTank has a separate chamber at the bottom through which tap water can be circulated, and the Bandit has a removable cooling module.
The Panther™ Semi-Dry Electroblotter is Owl's contribution to the options available for semidry transfer. The Panther, equipped with a stainless-steel cathode electrode and a platinum-plated titanium anode electrode, is available in two sizes: 20 x 20 cm and 35 x 45 cm. The latter, one of the largest semi-dry blotters available, is designed for sequencing gels or up to eight minigels--a useful capacity if you're interested in high-throughput blotting.
GSI is another manufacturer of semidry transfer systems, offering two models of the Investigator Graphite Electroblotter System: Type I for blots up to 18 x 18 cm and Type II for blots up to 24 x 30 cm.
To ensure maximum sensitivity and flexibility in your blotting application, it is important to choose the appropriate membrane for the job. A number of membranes are available for use in the transfer of proteins. Choice of membrane is dependent on a number of factors:
- Strength. Membranes differ considerably in chemical resistance and strength, important factors if the membrane is to be subjected to Edman degradation chemistry or is to be reprobed multiple times.
- Pore size. A pore size of 0.45 µm is recommended for most analytical protein blotting applications. For transfer of proteins with a molecular weight below 15,000 daltons, smaller pore sizes such as 0.2 µm are recommended. Retention of low molecular weight samples increases with reduced pore size, but transfer efficiency decreases when pore sizes below 0.1 µm are used.
- Binding capacity. High binding capacity increases the possibility that rare low-abundance proteins will be retained for detection after transfer.
- Type of detection method. Some membranes are better suited to a particular detection method than others, resulting in lower background and higher sensitivity.
Nitrocellulose, or cellulose nitrate, is a commonly used, hydrophilic, multipurpose transfer membrane. Protein binding to nitrocellulose occurs largely by hydrophobic and electrostatic interactions. Nitrocellulose membranes are available as 100 percent nitrocellulose or as a nitrocellulose-cellulose acetate mix, with the former having a higher protein-binding capacity. Most protein samples will bind strongly to nitrocellulose in low ionic strength buffers without subsequent treatment of the blot; however, some proteins require fixation onto the membrane by glutaraldehyde treatment or air drying. Nitrocellulose also offers the advantage of displaying low backgrounds (low nonspecific binding) for colorimetric and chemiluminescent substrates.
One of the disadvantages of nitrocellulose is its low strength. Most nitrocellulose membranes do not stand up to the rigors of multiple stripping and reprobing, which lead to discoloration and disintegration or cracking of the membrane. Supported nitrocellulose is produced by casting layers of nitrocellulose on an inert synthetic support, resulting in a membrane with the same binding properties as nitrocellulose but with superior handling characteristics.
PVDF binds proteins via strong hydrophobic interactions, resulting in higher binding capacities than nitrocellulose. This higher binding capacity virtually eliminates the "blow-through" effect, where low molecular weight proteins pass through the membrane and are not retained.
Because initial coupling yields are double those obtained with other materials, PVDF is especially suitable for transfer of proteins to be subjected to protein sequencing analysis. In proteome analysis, 2-D gel transfers are initially stained to determine protein location. The chemical stability of PVDF--it is virtually chemically inert--allows use of a range of solvents for rapid destaining and ensures that the membrane is compatible with the harsh chemicals subsequently used in Edman chemistry. Perkin-Elmer manufactures advanced PVDF membranes specially designed for protein transfers to be used in protein sequencing. The Blott system (ProBlott and Mini ProBlott membranes) permits lower backgrounds and improves sequencing sensitivity for a variety of proteins and peptides that are electrotransferred from gels.
The mechanical strength, chemical resistance, and high binding capacity of PVDF make it ideal for a variety of applications including Western and dot blotting. It can be used in most nitrocellulose protocols with the addition of a simple prewetting step. The unique properties of Millipore's Immobilon-P dramatically reduce the number and length of washes required during detection, a feature that promises significant time savings and reductions in nonspecific binding.
Though most companies recommend neutral or positively charged nylon membranes for use with nucleic acids only, Schleicher and Schuell does recommend its range of 0.2 µm pore-size Nytran® membranes for some protein applications. Nylon membranes offer mechanical strength and high binding capacities--in the region of 400 µg/cm2. The recently developed Nytran® SuperCharge has the highest positive charge of any available charged nylon membrane and promises maximum retention of small fragments and optimum sensitivity with isotopic detection systems.
It often seems that many of the steps in Western blotting are too short to allow you to achieve anything useful in the meantime, but everyone knows a watched blot never stains! Although many steps are involved, they are all relatively low tech, so it makes sense to automate the whole procedure. How nice it would be to load the relevant reagents into an automatic processor and come back when the procedure is done. Well now you can. Not only does automation speed up the procedure, it also promises to enhance reproducibility. We found three automatic processors on the market--all notable for their simplicity. Each of the processors is basically a liquid delivery and removal system. Receptacles are provided for loading reagents, and pumps are used to deliver reagents to the blotting tray and to aspirate reagents after each step.
Stovall's Washing Machine consists of four syringes to hold antibodies and other reagents and includes 4 L bottles for wash buffer and waste. If more than four reagents are to be used, additional steps must be performed manually before or after the processor is turned on. For overnight applications, the Cold Zone, an insulated box, maintains reagents at 4°C for over 20 hours. Reagents can also be returned to the Cold Zone after use, a useful feature when dealing with expensive or hard-to-come-by antibodies. Forty-five stored programs in the ROM automate procedures, and additional software is available for storage of up to 90 custom programs. A shaker platform is not included--the Washing Machine is designed for use with any shaker platform. Sample trays for large gels/blots and minigels/blots are provided, and for small-volume applications a clip is available that fastens the reagent and aspiration ports to any container, making the Washing Machine versatile enough for most washing or staining protocols.
Bio-Rad's Western Processor accepts reagent bottles of various sizes and can be used to deliver and remove up to six different reagents at user-defined intervals. Although the Western Processor doesn't include a cold-storage box, the equipment is cold-room compatible. A built-in rocker platform features three adjustable speeds for agitating blots, and trays are provided for both large blots and miniblots--two miniblots or one large blot can be processed at one time. For maximum reproducibility--one of the key features offered by automatic processors--Bio-Rad has put together a number of Western blotting kits called Immun-Blot Assay Kits (see table), for use specifically with the Western Processor. The Western Processor is preprogrammed with five optimized miniblot methods for use with these kits, virtually eliminating variation resulting from inconsistencies. Up to 10 additional user-defined protocols, each containing up to 15 steps (multiple washes can be considered as a single step), can be entered into the instrument's memory.
The new Processor Plus from Amersham Pharmacia Biotech has a variety of protocols preprogrammed for Western blotting, including ECL kits. Minimal user intervention is needed to run four mini or two standard blots for an ECL detection protocol. One can choose automated antibody delivery or manual addition and recovery of precious antibody. There are nine reagent ports for your choice of chemistry.
For diagnostic use, the CodaXcel™ from Immunetics, Cambridge, Mass., is designed to perform a Western blot in just 15 minutes, although it is designed to be used only with Immunetics CodaXcel test kits, currently available for HIV-1/2, HTLV, Chagas/Leishmania, and Lyme. Unlike the other two processors, the CodaXcel uses a vacuum to aspirate samples and reagents through the membrane, resulting in an enhanced reaction time between antigen and antibodies, greatly reducing incubation times. Although the currently available applications of the CodaXcel make this instrument primarily useful for rapid diagnostic procedures, particularly in a field setting, expect future developments in this technology.
Western blotting kits offer several advantages over purchasing reagents individually. For many people, the convenience of purchasing a prepackaged kit outweighs any additional cost that may be incurred, but perhaps the primary advantage of kits is that all the reagents have been optimized to work together at specific, predetermined concentration ranges. Additionally, reproducibility is often enhanced by eliminating the need to premix reagents. However, customers must still optimize reagent concentrations when it comes to enzyme conjugates. Some of the kits also include proprietary blocking reagents, buffers, and enhancers, further ensuring optimal performance with the combined kit components. For more sophisticated experimental design, some users may prefer the flexibility offered by purchasing individual reagents.
Most of the available kits rely on enhanced chemiluminescent detection (as introduced by Amersham Pharmacia Biotech) using an enzyme-conjugated secondary antibody (primary antibodies are not supplied) or enzyme-conjugated streptavidin (for detection of biotinylated proteins). In a chemiluminescent reaction, light is emitted from a substrate as a result of an enzyme-catalyzed reaction. In Western blotting, antibodies labeled with horseradish peroxidase (HRP) can be detected by the action of the enzyme on cyclic diacylhydrazides, such as luminol, in the presence of hydrogen peroxide and a chemiluminescent enhancer. HRP-catalyzed oxidation of luminol results in an excited product that decays to the ground state by emitting light. An HRP based signaling system has the advantage of generating a signal at a much faster rate than the alternative alkaline phosphatase system. However, signal intensity and duration is dependent upon the enhancer used.
Amersham Pharmacia Biotech's ECL® reagent was the first commercialized chemiluminescent substrate. This substrate uses a phenolic enhancer to generate light immediately on reaction with the HRP (L.J. Kricka, G.H.G. Thorpe, T.P. Whitehead, United States Patent 4,598,044, 1986). Other commercial luminol systems have been developed based on this same technology (NEB's LumiGlo® and DuPont's Renaissance system and S&S). A phenolic enhancer, such as 4-iodophenol, acts as a radical transmitter between the formed oxygen radical and luminol, increasing the resulting light emission by as much as 1,000-fold. Using luminol and 4-iodophenol, less than 10 picograms of antigen can be detected using X-ray film exposure times ranging from a few seconds to one hour. By capturing the light response on X-ray film, results are stable indefinitely and can be quantitated using densitometry. Alternatively, the use of charge coupled device (CCD) imagers can capture the light for easy quantitation, although longer exposure times are required per blot. Because the method is nonradioactive, it is not associated with any safety concerns or expensive waste disposal.
Comparison of different chemiluminescent POD substrates. Dilutions of ß-galactosidase (1 ng to 1 pg) were blotted after electrophoretic separation onto PVDF membranes, incubated with 3 mg/ml Mab against b-galacatosidase, and 50mU/ml anti-mouse IgG-POD. The blots were exposed for 10 minutes immediately following incubation with substrate. Courtesy of Roche Molecular Biochemicals.
Phosphate-containing dioxetane compounds, such as CSPD®, are widely used as chemiluminescent substrates for alkaline phosphatase conjugates and result in significantly lower background luminescence than first-generation 1,2-dioxetanes. CDP-Star®, an ultrasensitive chlorosubstituted 1,2-dioxetane substrate for alkaline phosphatase (patented and manufactured by Tropix but now available from a number of companies), is the fastest and most sensitive chemiluminescent substrate for membrane-bound alkaline phosphatase detection. It provides a five- to 10-fold brighter signal intensity than CSPD substrate. Exposure times resulting from incubation with CSPD substrate can range from 30 seconds to 45 minutes and with CDP-Star from 1 second to 30 minutes. However, these exposure times can only be achieved after allowing the enzyme's kinetics to reach optimum signal strength, which is 30 minutes to 1 hour after incubation with the alkaline phosphatase substrate. Glow kinetics associated with chemiluminescence of 1,2-dioxetane substrates, ensure a stable signal for up to 24 hours.
To increase signal intensity, a third layer can be added to the detection format. By using a biotinylated secondary antibody followed by a streptavidin-enzyme conjugate, as in the Western-Light Plus™ Kit from Tropix. Be aware that greater signal intensity with an alkaline phosphatase signal can also result in higher backgrounds. When the signal-to-noise ratio is reduced, the level of sensitivity also diminishes.
Bio-Rad's Western Blot Processor
Amersham Pharmacia Biotech's ECF Western blotting system includes fluorescein-labeled secondary antibodies, which are detected directly by a fluorescence scanning system (such as Molecular Dynamics' Storm™), avoiding the additional steps of exposing and processing X-ray film. This system is recommended only for detection of abundant proteins. For sensitivity comparable to that achieved using radioactive detection, the ECF Western blotting kit includes a tertiary antibody--antifluorescein conjugated with alkaline phosphatase--and an alkaline phosphatase substrate, AttoPhos®. AttoPhos was developed by JBL Scientific, San Luis Obispo, Calif., and is also available from that company. The precipitating product of AttoPhos cleavage generates highly fluorescent bands that can be readily quantitated.
Several manufacturers have tweaked the fundamental reagents used in Western blotting and detection to produce products promising enhanced results. Adequate blocking--a preliminary step designed to coat the membrane with protein at all sites where no antigen is bound--goes far toward eliminating nonspecific binding. Different antibody systems require different blocking reagents to provide the highest signal-to-noise results with either colorimetric or chemiluminescent detection. Several companies offer proprietary blocking buffers, such as ICN's Aurora™ blocking reagent, Pierce Chemical's SuperBlock® Blocking Buffer, and Schleicher and Schuell's Rad-Free Blocking Powder. It is also recommended to dilute antibodies and enzyme conjugates in these blocking buffers to produce the best results.
Many of the substrates used for colorimetric detection are unstable and must be prepared fresh for each experiment. Pierce Chemical's Metal Enhanced 3,3'-diaminobenzidine (DAB) Substrate for HRP requires that two components be mixed prior to use to ensure improved sensitivities compared with a 3,3',5,5' tetramethylbenzidine (TMB) substrate system. Many companies such as KPL, Bio-Rad, Roche, and Pierce supply stable, ready-to-use, one-component colorimetric substrates for both HRP and alkaline phosphatase. Promega's stabilized BCIP/NBT susbtrate for alkaline phosphatase, Western Blue® Stabilized substrate, promises to save time and enhance reproducibility. Opti-Membrane™ Reagent from ICN Pharmaceuticals enhances the signal obtained in chemiluminescent assays in both nitrocellulose and PVDF membranes, permitting shorter exposure times. It is a component of the Aurora™ Western Blotting Kit (see table).
Nitrocellulose membranes provide an inefficient environment for some alkaline phosphatase chemiluminescent systems, resulting in low signal intensity. Tropix has developed Nitro-Block and Nitro-Block II membrane enhancers to optimize the performance of 1,2 dioxetane substrates on nitrocellulose membranes by generating a hydrophobic environment on the membrane surface. Although it is not necessary to use Nitro-Block with hydrophobic PVDF membranes, it can result in 10-fold faster exposure times.
No matter which Western blotting system you use, empirical testing for the appropriate blocking buffer, antibody concentration, and required detection level is critical for survival on the wide-open plains of Western blotting.
One part of our Western blotting tale that has not been covered are the advances that have been made in recording signals generated on membranes. Improvements made in photographic equipment, densitometry computer programs and other signal recording devices can make your results look even better for that Gold Rush to publish. But we'll save that tale for another night around the LabConsumer campfire!
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