My downstream experiments require high protein concentrations from various cell lines, mostly, breast cancer cell lines. Here's my approach:
Briefly, I prepared 15 cm plates with 90–95% cell confluency, replaced the media with serum-free media, and collected the media, after 24 hours. The media then was spun down, filtered through a 0.22 nm filter, and concentrated using a 5 MW concentrator at 4000 g for 90 minutes. Despite these steps, the highest protein concentration I've got was around 2 mg/ml in only two cell lines. The rest were even lower. Any suggestions to further increase the concentration?
To obtain a higher amount of protein, you should increase the number of tissue culture flasks, as this will allow for a greater number of cultured cells to grow and produce protein.
Thank you. I’ve tried that. I even used 3 15cm plates. Still too low.
Okay. since you are collecting the media after 24 hours, extending the collection time to 48 or 72 hours might allow for more protein to be secreted, especially if the cell line has a slower secretion rate.Also check whether the serum-free media formulation is optimal for your cell lines. Some cell lines may require specific supplements or additives to enhance protein extraction. Additionally sometimes reducing the incubation temperature (e.g., from 37°C to 33°C) can reduce the degradation of secreted proteins and increase the protein yield.
These were quite useful suggestions. Thank you very much. Do you think adding protease inhibitors to the serum-free media could be useful? I checked the protein concentration at 20, 40, and 60 minutes, but the concentration remained low even after 60 minutes. So, I was wondering if adding protease inhibitors when switching to serum-free media might help.
You are welcome Dr.
Yes, adding protease inhibitors to your serum-free media could be a useful strategy. When transitioning to serum-free conditions, cells often release various proteases that could degrade proteins of interest, especially if the culture media lacks the inhibitory factors present in serum. By adding protease inhibitors, you would reduce protein degradation and help maintain the integrity of the proteins you're measuring.
If the protein concentration remains low even after 60 minutes, it could indicate active degradation, so using a cocktail of protease inhibitors (targeting both serine, cysteine, and metalloproteases) can be particularly beneficial to preserve protein levels for your analysis.
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