Big Problems with Novagen´s HisTag Protein Purification
Thorsten.Schmidt at rz.ruhr-uni-bochum.de
Sat Nov 9 11:03:40 EST 1996
Dear Sir or Madam!
I have big Problems with Novagen`s HisTag Protein Purification-Kit.
I use the pET30-Vector which allows translation of my target protein
6 His-residues. The target protein expression is inhibited during cell
induced by IPTG addition.
The 6 Histidines are used for protein-purification:
The Histidines bind to a "His bind resin" carrying Ni ions.
One should get 50-100 mg total protein after cell lysis and -after
purification- 20 mg target protein from one 100 ml culture.
But which differences between theory and my experience!!!
I followed strikly Novagen´s purification protocol but the problems begin
total protein. Instead of the predicted 50-100 mg, I get only 2 (!).
But there might be no fault possible: One just harvests the cells by
centrifugation, resuspends the cells in a buffer (containing 5 mM imidazol,
0,5 M NaCl and 20 mM Tris-HCl pH 7,9), sonicates and then removes debris by
ultra centrifugation (39.000 g).
Changes in the protocol (addition of Lysozyme, 0,1 % Triton) increases the
total protein amount only to 30 mg.
This leads to my first question:
What can I also do to increase the total protein yield?
May Lysozyme or Triton lead to problems with the HisTag purification?
The second problem is the colomn chromatographie:
According to the protocol I expect a flow rate of 25 ml/h.
But the real flow rate is just 10 ml/h. Filtration of the cell lysate or
new packing of
the column did not help.
Although SDS-PAGE analysis showed that the target protein is expressed, I
got no target protein (0,04 mg instead of 20 mg!).
Have you made experiences with this purification kit?
Why is the total protein concentration so low?
How can I increase the column´s flow rate?
Why is it impossible to purify my target protein?
Can you help me?
Thank you for your answer!
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