Electrophoresis/Western Blotting

Although the Western blot is a core lab procedure, surprisingly little has changed in traditional bench work practice since its inception in 1979. The V3 Western Workflow delivers a streamlined solution to the conventional western blotting workflow as explored here by one protein researcher specializing in chronic obstructive pulmonary disease (COPD).

Bio-Rad has long demonstrated innovation in imaging, with the introduction of the stain-free gel electrophoresis workflow, which was rapidly followed by the launch of the Gel Doc™ EZ imager. Now, this progression continues with the release of the ChemiDoc™ MP imaging system.

Hexapeptide libraries, such as Bio-Rad’s ProteoMiner™ protein enrichment technology, can significantly increase sensitivity of shotgun proteomics analyses of diverse complex samples. Underlying this increased sensitivity is the partial removal of high-abundance proteins leading to an increased representation of low-abundance proteins — a phenomenon described as dynamic range compression. Currently, hexapeptide libraries have been mostly used to increase proteomic detection in fluid samples (eg: saliva, plasma, etc.), but not in cellular lysates.

Today many researchers are considering changing their western blot detection method from chemiluminescence to multiplex fluorescence. There are several drivers behind this trend. Most significantly, fluorescent detection allows users to multiplex their western blots, enabling simultaneous detection of several target proteins at once, reducing or eliminating the need to strip and re-probe. Other benefits of fluorescence include better dynamic range, more quantitative results, and better signal stability over time.

2-D electrophoresis is a multistep technique that separates proteins first by a protein’s isoelectric point and then by its molecular weight. This powerful technique can separate thousands of proteins in a complex proteome and is useful for applications such as protein profiling and biomarker discovery. The drawback of the technique is that it requires multiple steps, each of which can be time-consuming and can contribute to problems with reproducibility.

This tech report compares in-gel and off-gel separation of proteins in terms of overall speed and simplicity of the workflow, resolution of the separation, and depth of proteome coverage provided. Results found that as well as higher peptide yields, in-gel separations were better suited to take full advantage of downstream analysis methods.

Mini-PROTEAN® TGX Stain-Free™ and Criterion™ TGX Stain-Free™ precast gels, when combined with Bio-Rad’s ChemiDoc™ MP or Gel Doc™ EZ imagers, provide researchers with the fastest protein separation and visualization available using the standard Laemmli system.

The ChemiDoc MP imaging system enables stain-free operation and lets you visualize proteins at every stage of your experiment. Its flexibility and sensitivity are complemented by a simple, intuitive operation that integrates seamlessly into your workflow.

Introducing Bio-Rad’s PROTEAN i12 IEF system. Each channel in the i12™ focusing tray is powered by its own power supply, enabling precise control over each IPG strip. This makes it possible to run different sample types, different gradients, and multiple protocols all at the same time.

This tech report demonstrates the advantages of independently controlled voltage and current parameters for each focusing tray in optimizing IEF conditions for a complex, insoluble membrane protein fraction.