1 reply to this topic

[silvepa23]

Hi,

I am expressing a his tagged protein in e coli.  I wanted to know if you could suggest whether or not i should use sonication.  i can lyse the cells under denaturing conditions since i will have to refold anyway.  Is it true chemical lysis provides more purity and butter yield.  could you suggest a lysis buffer, wash buffer, and elution buffer.  i beleve the wash buffer should contain triton and all buffers should have imadiazole.  thanks for any help.  all the best

[protolder]

silvepa23, on 26 June 2011 - 06:11 PM, said:

Hi,

I am expressing a his tagged protein in e coli.  I wanted to know if you could suggest whether or not i should use sonication.  i can lyse the cells under denaturing conditions since i will have to refold anyway.  Is it true chemical lysis provides more purity and butter yield.  could you suggest a lysis buffer, wash buffer, and elution buffer.  i beleve the wash buffer should contain triton and all buffers should have imadiazole.  thanks for any help.  all the best

Hola, sonication es a good method to broke bacteria,before denaturalization eliminate soluble proteins by strong centrifugation 5oKg 20-30 min and after, disolve the pellet in your denaturant agent.check a pH a bit far away of the isoelectric point of your protein. after solubilization centrifugue again and load the column.buffers? 25mM phosphate,0.5M Na Cl and 5mM mercapto ethanol for sonication, the same buffer with urea or HCl.guanidine and 10-20 mM Imidazole for the column. and the same with 500 mM imidazole for elution. Two more things be sure that your protein or a part of it isn´t in the first soluble supernatant of sonication,because  refolding could lead to a very low proportion of well folded protein. for that is better to study expression and induction to have any part of your protein soluble and well folded. Buena suerte