Hello.
I am working on heterologoulsy expressing and purifying a protein using the Bac-to-Bac method. The protein expresses as verified by western blotting. I typically do a 4.2 L expression using P2 baculovirus. I lyse the cells via sonication in the following buffer (5 mL per gram of wet cell mass): 20 mM Tris pH=8, 500 mM NaCl, 10 mM imidazole, 5% glycerol, DNAseI, and protease/phosphatase inhibitor tablets. After sonication, I centrifuge the lysed cells for 1 hour at ~54,000 x g. After centrifugation the supernatant is not viscous and has a clear yellow tint. This clarified supernatant will clog the IMAC column (I have tried both HisTrap and self poured columns). Only when I filter all of the clarified supernatant using a 0.22 um syringe filter can I load without clogging. Does anyone know a better way to solve this issue of column clogging?
No, you must filter the sample, even after centrifugation. I recommend filtering the sample just before feeding it to the column. Precipitation of material in the sample will always occur upon sample refrigeration.
Agreed. You always need to filter your lysate before loading, especially for an HPLC system, even after centrifugation. The centrifugation step removes large debris and clarifies it enough that it can be filtered.
Thank you for your feedback Omar Gonzalez-Ortega & Theresa Smith
Since you both say that filtering through a 0.22 um filter is required, what brand or type of filter are you using? The current methods/devices I use to filter are wasteful and time consuming. For example, Im currently using CellTreat syringe filters, PES 0.22um 30 mm diameter (#229747) to filter my clarified lysate. The filters clog after passing ~5 mL of clarified lysate through them. If I have 200 mL of clarified lysate it takes about 40 of the syringe filters. Alternatively, I have tried to use 250 mL PES 0.22 um filters (Celltreat #229706), but these clog quickly.
We'd just use 30 mm PES syringe filters (I think from VWR but I don't know that the brand matters). I wish I could say I knew of a better way to do it because they do clog ridiculously fast and need to be constantly switched out, but we were never able to find one. The best we managed was that if you filter it through .45 um syringe filters before the .22 um, then it goes a little faster and uses slightly fewer filters. If you can find filters with a larger surface area, that may also help.
All filters suck, for large volumes use lateral ultrafiltration. I do not filter lysates after centrifugation (I centrifuge at 21000xg for 2 min), I just centrifuge the sample before injection, but I use a column packed by me, such that I can change the inlet filter when I see that the pressure increases too much. It is easier and faster to change that filter for a clean one, after several sample injections.
Thanks again for your feedback Theresa Smith & Omar Gonzalez-Ortega
Omar Gonzalez-Ortega , what type of columns and column filters are you using?
I still have columns produced by Pharmacia, I really do not know if GE produces equivalent columns.
What beads are inside your columns? If clogging is a problem use bigger beads. Due to the lower surface area compared to the smaller beads the yield may be lower, this bigger beads are better suited for viscous solutions.
Sebastian Lühn I have used the cytiva FastFlow crude 5-ml HisTraps. Even these columns become clog when passing clarified sf9 cell lysate. As of now in my protocol I use a salt active nuclease rather than the commonly used DNase. This seemed to help during my last purification. From what I have read the typical DNA nucleases become inactive in NaCl containing buffers. I was able to pass more volume of cell lysate through a 0.22 um filter when I used the salt active nuclease. It didn't solve the problem, but it did help.
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