Posts Tagged ‘western blotting’

Semi-Dry Protein Transfer Explained

 :: 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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Protein Transfer Methods

 :: 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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Selecting a gel for western blotting

 :: Posted by American Biotechnologist on 10-12-2011

In today’s western blotting tip, we will look at how to select the appropriate gel.

Figure 1: Comparative separation of TGX Any kD acrylamide gel and 4-20% gradient gel. An E. Coli homogenate (20 μg) was separated on both Criterion™ TGX Any kD Stain-Free™ and 4-20% gels at 300V in 18 min, and the total protein content was visualised by Stain-Free detection using the Gel Doc™ EZ imaging system.

For a good separation of a complex mixture of proteins over a wide range of MW, it is usually recommended to use a gel that has a gradient of concentration of acrylamide across its length. Bio-Rad offers, within its new Mini-PROTEAN TGX™ gel line, a special flavour that extends even further the resolution between 10 and 100 kD, where most of the proteins separated in electrophoresis are present. Even with a homogeneous acrylamide %, its special chemistry generates this particular pattern. This Any kD gel represents a good choice for the optimal separation in that range. See in Figure 1 the comparative resolution of an E. Coli homogenate separated in a TGX Any kD and a 4-20% gradient gel. Note that this special gel, like all the TGX gels, has a 12 months shelf life, is compatible with the standard Laemmli Tris-Glycine-SDS running buffer and can run faster down to 10 minutes for the mini format.

Bottom line: unless you require a very tight resolution, your best bet is usually to pick a gradient gel. Why settle for looking at a narrow range of proteins when you can have good separation of many proteins in one gel?

Western Blotting Tutorial Series on American Biotechnologist

 :: Posted by American Biotechnologist on 10-11-2011

SDS-PAGE and western blotting are traditional technologies in most laboratories working with proteins, in order to separate, visualize and identify some proteins within a mixture. A general detection of all the proteins can be done directly within the electrophoretic gel. The most commonly used protein blotting technique, western blotting, was developed as a result of the need to probe for proteins that were inaccessible to antibodies while in polyacrylamide gels. Western blotting involves the transfer of proteins that have been separated by gel electrophoresis onto a membrane, followed by immunological detection of these proteins. Western blotting combines the resolution of gel electrophoresis with the specificity of immunoassays, allowing individual proteins in mixtures to be identified and analyzed.

In the coming days, we will provide you with some helpful tips and tricks for achieving spectacular western blot results.

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Improving the statistic significe of your Western Blots

 :: Posted by American Biotechnologist on 09-08-2011

Western Blotting is probably one of the most ubiquitous techniques in the molecular biology lab and relatively easy to perform. Yet many of us have been frustrated with statistically insignificant results or protein bands that appear either too dark or too light to quantitate.

Well, do not despair! There are many things you can do to help improve the quality of your blots and increase your likelihood of obtaining statistically significant results!

In the video below, you will learn about the many factors affecting western blot analysis (such as detection limit and dynamic range limitations of film and overloaded gels) and what can be done to improve your chances of success.

The presentation was given by Bio-Rad Laboratories Field Application Specialist Dr. Sean Taylor as part of an intimate customer training. Some of the references in the presentation may be specific for that particular customer but the general information contained in this presentation is highly valuable to all molecular biology labs.