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Archive for the ‘Western blot tutorial series’ Category

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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