Hi All,
I worked with his tagged protein (37KD). It is soluble protein after overexpression of pet28a in Rosetta cells. At that time everything was fine. I got purified protein with 150mM imidazole but not now. My protein can be seen on SDS page gel with 20mM, 50mM imidazole but not pure ones. Does it mean my protein cannot bind in Ni-NTA? Resin problem? -----may be not because I changed new resin. Rosetta glysterol stock? Hopefully, you all kindly suggestions. Thanks in advance !
Hi,
Firstly please don't make glycerols stocks in B strain cells (including Rosettas), they are not recA etc. so can potentially modify your plasmid! Always keep a glycerol in a good cloning strain and re-transform miniprep into your expression strain not more than one week before you want to grow expression cultures. This is much better practice and generally gives much more reliable expression results and means that you have a reliable glycerol stock of your plasmid.
Secondly, are you sure that it is your protein in the low imidazole washes? It would be very unusual for your protein to be here, if it is here then it is more likely that you have a problem with buffers-e.g. wrong imidazole concentration, low pH, reducing agent (depending on resin) than a change in your protein. Your resin should be OK for at least 3-4 uses if it is properly washed or regenerated (again depends on which resin you use).
As above, it is important to wash and regenerate your resin. If it still contains imidazole from the previous purification, your His-tag has nowhere to bind to.
Hi Nick and Barbara,
I appreciate for great suggestions. I hope it helps.
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