Big Problems with Novagen´s HisTag Protein Purification

[Dima Klenchin]

In article <01bbce57$8fc130a0$09019386 at Schmidt.rz.ruhr-uni-bochum.de>, "Thorsten Schmidt" <Thorsten.Schmidt at rz.ruhr-uni-bochum.de> wrote:
#Dear Sir or Madam!
#
#I have big Problems with Novagen`s HisTag Protein Purification-Kit.

#One should get 50-100 mg total protein after cell lysis and -after
#purification- 20 mg target protein from one 100 ml culture.

#I followed strikly Novagen╢s purification protocol but the problems begin
#with the
#total protein. Instead of the predicted 50-100 mg, I get only 2 (!).

Did you have 0.5-1 mg/ml to start with? Not all proteins are expressed in 
soluble form to this level. 

#The second problem is the colomn chromatographie:
..
#How can I increase the column╢s flow rate?

1) use "real" chromatography with peristaltic pump
2) connect tubing to the outlet of you gravity column; the lenth of tubing 
will control flow rate
3) [maybe] dilute you lysate 

- Dima