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:: 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?
:: 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.
:: Posted by American Biotechnologist on 07-18-2010
What do you think of when I say the words electrophoresis and gel imaging? Are you jumping up and down screaming, YEESSSS! Let me have at those gels!? Or perhaps a subdued yawn is more up your alley? If you’re anything like me, you understand that protein electrophoresis and Coomassie staining (or whatever other stain you like to use to visualize your protein bands) is a necessary evil of most protein experiments. All we really want is to get to our results. Upregulated, downregulated, phosphorylated…whatever-ated. However, we all know that HOW we get our results is just as important as the results themselves. In fact, the “how” will often have a profound effect on the experimental outcome.
Although protein electrophoresis and visualization is a staple technique in any proteomic protocol, it tends to add a significant delay in our time-to-results. Typically, protein electrophoresis is performed at 200V for 1hr followed by a 1hr stain, a 1hr to overnight destain and a 30min to 1hr imaging process. Altogether, this presents anywhere from a 4hr to 24hr bottleneck in our experimental process. This is why I get very excited whenever a technology is introduced that can shave some time off of this time-consuming technique.
Bio-Rad Laboratories’ Criterion Stain Free Imaging System coupled with the new Criterion TGX Stain Free Gel enables you to run your gel much faster than the standard protocol (20min at 300V as opposed to 1hr at 200V) while relieving you of the need to stain and destain the gel prior to imaging. The entire process from gel run to imaging takes approximately 30 minutes as opposed to 4-24hrs! That’s a HUGE time savings. Moreover, the whole process is compatible with downstream applications such as western blotting and mass spec analysis with imaging sensitivity that is at least on par with coomassie staining.
So how does the technology work? Bio-Rad’s Criterion Stain Free Gels contain a trihalo compound that reacts with tryptophan residues in the protein sample during gel electrophoresis. The gel is then placed in the Bio-Rad Criterion Stain Free Imaging system and, with the push of a button, a UV-induced chemical modification of the trihalo compound produces a fluorescent signal that is detected by the imaging system (Image Lab Software provides automated image analysis once the image has been captured by the Criterion Stain Free Imaging System). It is important to note that the Criterion TGX Stain-Free gels are stable for 1yr, can be run at high voltages (hence the 300V, 20min run) and utilize standard Laemmli buffer (so you can continue using your standard Tris-Glycine buffer to run these gels).
I’ve attached a few key pieces of literature including the system brochure and tech notes which showing the system’s mass spec and western blotting compatibility. There’s also a fun promotional video that Bio-Rad uploaded to YouTube which I’ve included below.