2D gel electrophoresis (2DE) is a key technique for purifying individual proteins from complex samples based on their isoelectric points and molecular weights. Simple enough in theory, but as the plethora of protocols and articles shows, 2DE demands patience and meticulous optimization. But whether your samples are human sera or HUVEC lysates, 2DE uses these four core steps: sample solubilization, isoelectric focusing (IEF), SDS-PAGE, and – whew! – analysis.
Step 1: Sample Solubilization
Much like regular ol’ SDS-PAGE, those tissue or blood samples for 2DE need to be processed and solubilized before they can be loaded into the IEF gel. In an ideal world, all proteins would solubilize immediately with no qualitative or quantitative changes. In the real world, not all proteins are equal, and this first step immediately tilts 2DE towards detecting highly soluble and abundant proteins. Don’t reach for the Tween and Triton, however; solubilization requires IEF-compatible lysis reagents, such as electrically neutral detergents (e.g. CHAPS) and chaotropes like urea. Obnoxiously enough, urea can carbamylate proteins, while those pesky detergents can interfere with downstream steps! As a result, this step requires a great deal of sample-specific optimization to avoid bias and contamination.
Step 2: Isoelectric Focusing
At last, your sample is solubilized! Time to load it on an IEF gel where, similar to SDS-PAGE, the proteins will be pushed through the acrylamide gel by an electric field. Where IEF gets more exciting is that the gel incorporates a pH gradient, and each protein moves only until it reaches its isoelectric point (pI). The pI is the pH where a protein has no net charge, meaning the field has no effect and the protein stays put, focusing tightly into a band within 0.01 pH unit of its pI. As straightforward as this sounds, IEF throws several wrenches into the scientific works. First, proteins become less soluble and can even precipitate out as they move closer to their pI, especially in low-salt, IEF-friendly buffers. Second, IEF gels and buffers interfere with sample prep for mass spectrometry (MS) and can be difficult to stain for analysis. This means IEF must almost always be done first so that SDS-PAGE can make the sample MS-compatible. Finally, it’s very easy to contaminate your sample with keratin, so this step requires gloves, diligent depilation, and working behind a “sneeze shield.”
Step 3: SDS-PAGE
Finally, the second dimension! But first, those beautifully-focused proteins need to be solubilized again in SDS before they can be separated by their molecular weights on an orthogonal second axis. Other than this equilibration and some additional care with timing and voltages, this step uses molecular weights to separate proteins through the tried-and-true SDS-PAGE method. At the end, the gel will have the proteins aligned along two axes: isoelectric point vs. molecular weight.
Step 4: Analysis, or, Step 1: Preparation for Mass Spec
This final step depends on your particular experimental endpoints: are you comparing protein expression? and/or identifying proteins? When comparing protein expression across different experimental samples, the gels are typically stained with silver or Coomassie blue for total protein. Various image analysis platforms are then used to scan and compare the location and intensities of the separated proteins. Naturally, analysis is not as clean and simple as slapping a gel into the scanner. To improve reproducibility between samples, gels should be run in parallel, which can lead to logistic and diplomatic challenges in a lab with only one power supply and an imminent lab meeting. Furthermore, interpreting that Morse-like pattern of dots and splotches requires protein identification and annotation beforehand, either through in-house efforts or via sweet, sweet annotated databases of similar samples.
There are two general options for those in the protein identification phase. The first is to run a western blot, transferring the proteins from the SDS-PAGE gel. The second approach – which is more powerful, laborious and expensive – is to excise the proteins in the gel, digest them, and send them out for identification by MS. While this can yield buckets and buckets of high-resolution information – for example, the type of post-translational modifications or disease-related adducts to proteins, amazingly enough! – there are stringent requirements for a sample to be MS-worthy. Clean-up steps are typically required to remove the detergents and protein stains, and some stains – notably silver, which often requires development with a formaldehyde solution – interfere with MS sample preparation. Furthermore, MS requires typically several hundred nanograms of protein per sample, which means your protein of interest must be visible by the less-sensitive Coomassie blue stain.
Are there any alternatives?
Desperate researchers have found several alternatives to 2DE. For purifying a target protein for MS analysis, an antibody-based bead separation system (e.g., Dynabeads) can be used if (big if!) the target is known and a monoclonal antibody available. Some scientists also replace the IEF step with a zymogram, but this requires working with a known enzyme and one of its established substrates.
2DE is an intimidating technique, but not a lonely one! If you are considering using this approach, I would recommend reviewing some of the references below, especially the annotated databases of specific samples run through 2DE. Time is precious, and there is no need to re-annotate the wheel (or at least, that HepG2 lysate).
In studies of RNA abundance and gene expression, no one technique can answer all of the questions that need to be asked. So it is necessary to use a variety of experimental methods in concert. Two RNA detection and measurement techniques that complement each other well for this purpose are RNA Fluorescence in situ hybridization […]
It’s great to have you in the Bitesize Bio family! We’ve sent you an email to confirm your registration. Please click on the link in the email or paste it into your browser to finalize your registration.
For more information on how to use Bitesize Bio, take a look at the following image (click it, for a larger version)
An error occured while registering you, please reload the page and try again