Hi all,
Recently, I expressed human NAT10 in Rosetta(DE3) at a 2 uM IPTG level (final concentration). I used 50 mL culture for this as I did for the first time (thinking to start with minimum amount of culture volume). After the induction at 16 C for 4 hours (100 rpm) I extracted the protein Ni-NTA (as the protein has TEV+SUMO+His tag) and ran an SDS PAGE. But, I did not able to see a band on the gel. Therefore, my questions would be like why I couldn't see a band on the gel? Do I need to increase the IPTG concentration? Do I need to increase the culture volume? So, appreciate all of your valuable suggestions to solve this problem. Thank you.
My lab usually uses a final IPTG concentration of 0.5 to 1 mM, so I think you can increase the amount of IPTG you use. 2 µM IPTG is too low. Also, you should optimize the induction conditions, such as using different temperatures (16, 25, 37 °C) and IPTG concentrations (0, 0.25, 0.5, 1 mM). Hope this helps
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