IDPs are proteins lacking globular structure and defy the classic protein structure-function paradigm, but in recent years, have been found to be integral in cellular regulatory pathways and protein interaction networks. Facilitating biophysical characterization of these proteins entails high purity of samples and the use of various chromatographic strategies.
Q: What kind of chromatography do you use? What is it used for?
A: We employ from low, medium, to high pressure chromatography systems. We have the new Bio-Rad NGC FPLC and other HPLC systems in the lab. We need a variety of chromatography methods and columns to purify different types of proteins or biomolecules depending on their unique biochemical or biophysical properties. We employ a variety of purification strategies, using affinity, ion exchange, size exclusion, and reverse phase chromatography. And, especially for intrinsically disordered proteins, we have to rely on denaturing methods where we totally unfold the proteins, because many of these IDPs are mostly insoluble when they’re over-expressed in E.coli.
Q: What is the first thing you tend to look at when something goes wrong with your chromatography systems? How do you progress from there?
A: First, I try to isolate the problem. With the NGC, the system is separated into different modules. Usually the pumps, the detectors, and the different components each have a sensor and a reading, so first it’s important to look at the manufacturer’s manual for the specific error codes. If the problem occurs during the chromatographic run, I look at the pressure. It’s the best gauge of what’s wrong with the system. We watch the baseline pressure with or without the columns and it’s also a good thing to keep a log of the pressures. We have a logbook of particular columns and how much pressure each one is going to hold, etc. With the NGC right now, they have already put a lot of the custom columns from different companies into the system, not just Bio-Rad, but also GE, ÄKTA, etc. They have the precise pressure limits, so it’s more customized and less error-prone when you’re running them. We also check the flow rates. So if the pumps are saying 5 mls per minute, the flow rate should be 5 mls per minute, and if it’s not, you have to check each section for clogs and leaks in the tubing and the connections. If the pressure is high, there might be clogs, like salt in the lines and in the connections or some air bubbles in the system. If there are air bubbles, then you need to purge the lines.
Q: How can you prevent such issues?
A: For maintenance, the pump seals and buffer or column filters or frits might need to be cleaned or changed. It’s important to do regular maintenance— you especially don’t want to leave the lines full of salts. We typically do regular maintenance—daily, weekly, and monthly. For example, after the runs of the day, we make sure that we wash the whole system with degassed filtered water or you can also put 0.01% azide with the water to prevent bacterial growth or 20 percent ethanol. We also do weekly or monthly cleaning of the lines with 0.01 to 1N NaOH or organic solvents. It’s also important to clean the columns, which can be done by reverse flow or manually turning the column upside down. A lot of times, there is a lot of gunk stuck on top of the columns, so you want to reverse them and then remove that gunk.
Q: What are the most common problems you run into with your systems?
A: Right now, it’s related to the run— pressure fluctuations, so high or low pressure that’s caused by air bubbles, clogs, or salts. It’s just about really knowing your system, really knowing the baseline pressure, with and without the columns, and making sure that there is no air in the system. If, for example, you suspect that it’s the column that’s clogged, then you remove the column or try a different column and see if the pressure goes back to normal. Then you know that it’s the column and you’ll have to clean it. If it’s a problem with the system itself then there might be leaks in the tubing or connections, or there are bubbles in the system.
Q: How has troubleshooting chromatography systems changed for you over the past few years?
A: Troubleshooting has definitely gotten easier due to advancements in technology and better software. For example, it’s now easier to see pressure readings in the software interface on the computer and it will report both pre-column pressure and the pressure change across the column. Before, you had to speculate and you only knew the pressure in the pump. You had to remove the column and do all sorts of things. Now you can see [right in the software] that it’s a problem with the column. Also, in the new FPLC systems, there are now air sensors so you can easily detect air in the system, and if the system detects air, the flow stops so it won’t introduce any air into the columns. The software is also much more informative, particularly with the new NGC. It tells you if there are issues in your system such as overpressure. You set pressure at a certain limit and if it goes above that limit, the system will automatically stop so you don’t ruin your column. You can also directly see the fluidics scheme in the software. If you push a particular button, you can directly see the effect on the fluidics in the interface and where the fluid is going. Another thing is you can bypass the columns without manually taking them out to know the pressures with and without columns. There’s the reverse flow technology, where you can reverse the flow in the column without manually inverting the column. So, with a touch of a button, you can easily clean your columns. The chromatographic systems have also gotten better at preventing issues.
Q: What resources for troubleshooting chromatography systems have you found to be the most useful?
A: First, I always go to the manufacturer’s troubleshooting guide, the error codes and so forth. But I also use the Web. There’s a lot of expertise out there, blogs and forums on how people deal with the different problems. You just have to find the solutions out there and try them to find what works for you.
Q: For lab professionals who are still learning or becoming familiar with chromatography systems, what are the most important things they should focus on when troubleshooting?
A: You really need to take the time to get to know your system. Especially now, when there’s a lot of ports, a lot of options for the way the fluid goes, they have to be really familiar with the fluidic scheme of the system, where the lines and fluid go, where your sample goes in, and so on. They have to know when they do one thing [in one part of the system], what happens to the other parts. The best things to focus on are the flow rate and the pressure. Make sure that the flow rate is the one you observe in the outlet and if it’s not, there are some leaks or clogs somewhere in the system. You have to really watch what the baseline pressure is without the columns.
Q: Did you have anything else you wanted to add?
A: To avoid troubleshooting, the best way to prevent problems is regular maintenance. It’s very important, along with keeping a log of changes in the pressure, and column uses. Regular cleaning of columns, using reverse flow, denaturants to dissolve insoluble proteins, and organic solvents, though with those you have to check the manufacturer’s manual for particular columns on their chemical compatibility and cleaning suggestions. Salts are especially nasty to the pumps and lines because they dry and harden and then clog up the system. It’s really important to flush your system with water after the run. Make sure that you filter your samples and degas and filter the buffers so there are no particulates. If you do all of this, you prolong your regular changing of the filters and you can extend the life of your columns and the life of the machine.