I've been trying to express eukaryotic phosphatase in E.coli bl21 de3 Plyss. I had initial success (30 mg) but now it seems that all I get is lac repressor when purifying the next batches. Growth conditions: one colony from fresh transformation plate to 100 ml TB, 37 until od 1.0, with 50 ug/ml Kan+ 34 ug/ml Chl. Inducing with 1 mM IPTG and o/n growth at +18. Using Maxwell His purification kit to check the expression levels since the protein has c-term His tag. But all I get is lac repressor that gets a reallyfat band. Any thoughts?
What should bind to the matrix is your his tagged phosphatase. Why do you think that it is the lac repressor (lac repressor is not his tagged).
Hi,
Try inducing with IPTG when the OD is between 0.45 and 0.60. I think your cultures are entering stationary phase. Do you make a growth curve to show your cells are growing exponentially? Also, 18 degrees Celsius is pretty cold. After we induce, we immediately turn the heater "OFF" on our shaking incubator. The following day the Temp. is usually around 30 deg. C.
Hope this helps.
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