:: Posted by American Biotechnologist on 01-30-2012
The most common method for analyzing protein expression levels is western blotting with detetion of a single protein target, using horseradish peroxidase-conjugated or alkaline phosphatase-conjugated antibody probes combined with colorimetric or chemiluminescent detection. While these methods work well for studying a single target, they are unsuitable for anlayzing multiple targets at the same time, particularly if the target proteins are of unknown or similar sizes. For analysis of multiple targets, the blot is typically stripped and reprobed for additional targets of interest. Reprobing is time consuming, and often some of the target protein on the blot is lost as a result of the stripping procedure. If one protein is removed to a greater of lesser extent relative to another protein, the ability to quantitate the relative amounts of diffferent proteins of interest is compromised.
In this technical note, you will be introduced to fluorescent western blotting detection which is superior to traditional western blotting when trying to analyze multiple proteins.
- fast and quantitative detection of multiple proteins in a single experiment
- sensitivity compared to chemiluminescent detection
- linear dynamic range up to 10 times greater than that of chemiluminescent detection
- fewer experimental steps than chemiluminescent detection
- no substrate requirement, and therefore no risk of exhausting the substrate and causing a “dead zone” in the blot
- the ability to visualize and quantitate both phosphorylated and non-phosphorylated forms of individual proteins
The technical note is divided into three sections to help those who are new to fluorescent western blot detection quickly generate reliable and reproducible results.
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:: Posted by American Biotechnologist on 10-17-2011
With its proprietary transfer buffer, the Trans-Blot Turbo system generates very fast transfer even for high molecular weight proteins. However, as indicated previously, the gel composition, i.e. the acrylamide – bis-acrylamide network density, influences the transfer efficiency. A protein can more easily move out of the gel during the transfer if it is located in a portion of gel that has the widest pore structure. As proteins above 150 kD are always located on the first top part of a gel, the most efficient transfer of those large proteins is achieved when using gradient gel with a concentration of 4% of acrylamide – bis-acrylamide at the top of the gel. The transfer efficiency of proteins from an Any kD homogeneous acrylamide gel and a 4-20% gradient gel is illustrated.
Qualitative transfer efficiency comparison of HMW proteins on homogeneous acrylamide % and gradient gel. Precision Plus Protein™ Unstained standard and E. Coli homogenate (20 μg) were run on both Criterion TGX Any kD Stain-Free and 4-20% gels at 300V for 18 min. The total protein content was detected with the Stain-Free technology using the Gel-Doc EZ imaging system. The gels were then transferred with the Trans-Blot Turbo system with the 7 min preset program using the Trans-Blot Turbo PVDF transfer packs. The total protein content remaining in the gel is detected with the Stain-Free detection, using the same exposure parameters as used with the gels before the transfer for reliable comparison. The content of protein was also detected with the Stain-Free detection on the membrane. Even if most of the proteins are transferred in 7 min, the gradient gel contains less HMW proteins than the homogeneous gel after the transfer.
:: Posted by American Biotechnologist on 10-16-2011
In theory, increasing the power input and duration of an electrophoretic transfer results in the transfer of more protein out of a gel.
In practice, however, test runs should be used to evaluate transfer efficiency at various field strengths and transfer times for each set of proteins of interest.
The optimum transfer conditions depend on a number of factors, including the size, charge and electrophoretic mobility of the protein, the type of gel and transfer buffer used, and the type of transfer system being used.
In a semi-dry transfer system, the distance between electrodes is determined only by the thickness of the gel-membrane sandwich, and buffering and cooling capacity is limited to the buffer in the filter paper. As a result, the field strength is maximized in semi-dry systems, and the limited buffering and cooling capacity restricts the transfer time. Though power conditions may be varied with the power supply, semi-dry transfers often operate best within a narrow range of settings.
:: Posted by American Biotechnologist on 10-14-2011
In semi-dry systems, the distance between the electrodes is limited only by the thickness of the gel and membrane sandwich. As a result, high electric field strengths and high-intensity blotting conditions are achieved. Under semi-dry conditions, some small proteins may be driven throughout the membrane in response to the high field strengths. Moreover, because low buffer capacity limits run times, some large proteins may be poorly transferred. Use of a discontinuous buffer system may enhance semi-dry transfer of high molecular weight proteins (>80 kD). As semi-dry transfers require considerably less buffer and are easier to set up than the tank method, laboratories performing large numbers of blots often favour them.
Novel buffer and material formulations have been developed that can be used with higher electric field strengths than those used in typical semi-dry blotting. These conditions yield complete and extremely rapid transfer, with some systems completing transfer in 3 – 10 min. Such rapid blotting systems do not incorporate external cooling mechanisms, so the high power dissipation may generate more heat than other techniques. Rapid blotting systems are intended for extremely rapid transfers where heat-induced protein denaturation will not affect downstream applications.
The Trans-Blot Turbo system performs semi-dry transfers in as little as 3 min. The system uses prepackaged transfer packs containing a prewet membrane (nitrocellulose or polyvinylidene difluoride:PVDF) and filter paper stacks soaked with a proprietary buffer that allows this fast and efficient transfer. The base unit contains an integrated power supply that drives two independent transfer cassettes, allowing transfer of a total of four mini-format or two midi-format gels. As the transfer cassette electrodes are made with robust permanent stainless-steel and platinum-coated titanium, the Trans-Blot Turbo system also has the flexibility to run traditional semi-dry protocols with hand-made buffers and any membrane, as any semi-dry system.
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:: Posted by American Biotechnologist on 10-13-2011
The protein blotting workflow involves selection of the appropriate gel, method, apparatus, membrane, buffer and transfer conditions. Once proteins are immobilised on a membrane, they are available for visualisation, detection and analysis.
There are two main types of electrophoretic blotting apparatus and transfer procedures:
- Tank transfer systems – gels and membranes are submerged under transfer buffer in tanks; these systems are useful for most routine protein work, for efficient and quantitative protein transfers, and for transfers of proteins of all sizes. Tank transfer systems offer the most flexibility in choosing voltage settings, blotting times and cooling options.
- Semi-dry systems – gels and membranes are sandwiched between buffer-wetted filter papers that are in direct contact with flat-plate electrodes; these systems are typically easier to set up than tank systems and are useful when high-throughput is necessary and extended transfer times are not required or when discontinuous buffer systems are used. Active cooling options are limited with semidry blotting.
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