Q: What are the most common problems you run into with your systems?Ī: Right now, it’s related to the run- pressure fluctuations, so high or low pressure that’s caused by air bubbles, clogs, or salts. 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. It’s also important to clean the columns, which can be done by reverse flow or manually turning the column upside down. We also do weekly or monthly cleaning of the lines with 0.01 to 1N NaOH or organic solvents. 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 typically do regular maintenance-daily, weekly, and monthly. It’s important to do regular maintenance- you especially don’t want to leave the lines full of salts. Q: How can you prevent such issues?Ī: For maintenance, the pump seals and buffer or column filters or frits might need to be cleaned or changed. If there are air bubbles, then you need to purge the lines. 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. 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. They have the precise pressure limits, so it’s more customized and less error-prone when you’re running them. 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. We have a logbook of particular columns and how much pressure each one is going to hold, etc. We watch the baseline pressure with or without the columns and it’s also a good thing to keep a log of the pressures. It’s the best gauge of what’s wrong with the system. If the problem occurs during the chromatographic run, I look at the pressure. 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. With the NGC, the system is separated into different modules. 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?Ī: First, I try to isolate the problem. 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. We employ a variety of purification strategies, using affinity, ion exchange, size exclusion, and reverse phase chromatography. 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 have the new Bio-Rad NGC FPLC and other HPLC systems in the lab. Q: What kind of chromatography do you use? What is it used for?Ī: We employ from low, medium, to high pressure chromatography systems. Facilitating biophysical characterization of these proteins entails high purity of samples and the use of various chromatographic strategies. 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.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |