When you think about separating proteins, do you think about separating them using a gel? Specifically using SDS-PAGE? If you answered “yes”, it is for good reason. SDS-PAGE is ubiquitous in molecular biology labs because it is good at separating proteins. However, SDS-PAGE takes a lot of time and is labor-intensive. So let’s expand your toolbox and look at another way of separating proteins: Capillary gel electrophoresis.
Capillary gel electrophoresis (CGE) a.k.a. capillary sieving electrophoresis a.k.a. SDS-capillary gel electrophoresis separates proteins in a gel via columns filled with a separating medium.
In capillary gel electrophoresis your analytes (the proteins you want to study) migrate through an electrolytic solution through capillaries by the pulling force of an electric field – not unlike the more familiar SDS-PAGE. To perform CGE you need: a sample vial, source and destination vials, sieving matrix, capillary column, an on-column detector, a data output and handling device and a high-voltage power supply.
Protocol in Brief
In a CGE experiment your sample starts at the source vial and heads towards the destination vial. Here is the protocol in brief:
Step 1: The source and destinations vials as well as the capillary connecting them are filled with an electrolytic solution and the sample in introduced into the capillary.
Step 2: Once the electric field is applied, the analytes migrate from the source vial side to the destination vial side.
Step 3: The on-column detector (not surprisingly) allows for on-column detection. The detector sends data to a data output and handling device, such as a computer.
Step 4: This data is then displayed as an electropherogram. Your analytes are read as peaks of various retention times.
To find more details on how this works visit UC Davis’ helpful material.
More About the Matrix
Your sieving matrix can be composed of a number of polymerized materials, including cross-linked polyacrylamide, dextran, and poly(ethylene glycol or ethylene oxide). Your columns are usually made from replaceable sieving polymers. It is then up to you if you make your own columns or buy them. Beckman Coulter, Agilent Tech and Bio-Rad Laboratories sell sieving kits but know that these require you to use their CGE instruments too.
Why You Should Use CGE
Now you’ve heard about what CGE is, why should you use it?
- It has a shorter operation times. Running a CGE takes 10–100 times less time to complete than slab gel electrophoresis.
- There are less steps in a CGE experiment than in a SDS-PAGE experiment. This is because the end results of CGE experiment is given by a computer connected to the apparatus.
- CGE is also better suited to small proteins than traditional SDS-PAGE.
- You can analyze your proteins in real-time with CGE.
- CGE equipment can be used repeatedly, no need to make endless gels.
- Lastly, much of the process is automatic. After you’ve added your sample, you’re done! This means you don’t have to supervise the process like you do a gel or bring it to special detecting equipment as soon as the gel has run.
Why You Should NOT Use CGE
Well, truth be told even though CGE has been around in its present form for over a decade and many technical advances have been made, CGE still suffers from reproducibility issues. Another problem is that CGE separations are performed in series. This means with CGE you cannot conveniently compare lanes. Lastly, when it comes to 2D gels, CGE just can’t compete with traditional 2D separation.
In summary, while some researchers are proudly exclaiming the uses of CGE technology, there are still a number of issues with it. But don’t dismiss CGE just yet! The latest push in this area is microchip CGE, which shows much promise for high-speed protein analysis. So next time you think about protein separation don’t forget CGE.
Have you tried CGE? How do you like it compared to SDS-PAGE?