Hey everyone. I am currently purifying a protein using a S200 10/300 GL size exclusion column on an Akta purifier. I injected the protein and the UV is going crazy. The protein was filtered and centrifuged at 10,000g for 10 minutes before injection, and I inspected the system for leaks and water bubbles, neither of which are present. As seen on the green line, the pressure is stable. So I don't think it's caused by air on the pumps.
Does anyone have an idea what this could be? Maybe air trapped in the UV meter or a dirty flow cell? Screenshot attached.
Thanks in advance!
Air or some denatured protein trapped in the flow cell is the most obvious possibility. Try taking out the flow cell and giving it a thorough cleaning by the recommended procedure. An old, dying UV lamp is another possibility.
I must sadly say that notorious FPLC is bad as compared to the HPLC. I have experienced this issue at The University of Tokyo, Yayoi-cho, Bunkyo-ku, Tokyo in 2009 - 2013, during my working period as an Arbeit person (my unpublished experience).
SEC-Protein analysis should be performed by HPLC-Surfactant-SEC method (please see file; SEC column 300A silica). Fucoidan (Sulphated poly-L-Fucose) has been safely determined using HPLC (please see file; JCB Fucoidan transport).
FPLC can not ustilize non-ionic surfactants such as Brij-58, and can only be used in low-pressure SEC mode. Surfactant in SEC is very important to give high recovery of proteins and other biological macro molecules. We have previously proposed to use non-ionic detergent NP-40 in order to prevent absoption of proteins to polyacrylamide gels (please see file; IEF for hydrophobic protein). High-recovery conditions uniquely and simultaneously prevent the troubles in HPLC analytical system including detection unit. Therefore, the recovery procedure by washing low-pressure FPLC system would require many and repeated times in Biochemical study.
By the way, Superdex Increase and/or Superdex columns are not possible to be re-packed by the biochemical researchers. On the other hand, HPLC columns are possible to repair the column-inlet portion and to be re-packed (please purchase my book; My Battle in Biochemical Biotin Study).
Adam B Shapiro I thoroughly cleaned the system with Guanidine Hydrochloride and reattached the column. The fluctuations are gone now. Thank you for the advice!
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