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[Seraph524]

Hi!

So I tried inclusion body prep from E Coli for the first time (used to always work with soluble proteins), and I think I did something wrong... When I lysed my induced cells and washed the inclusion bodies, my tech made mention that they weren't the expected "chalky white," but were kinda brownish and gooey. Additionally, after I washed them, I denatured the inclusion body pellet with 6 M G-HCl. The pellet immediately turned brown, and the next day after overnight denaturing, the solution was brown.

I proceeded and realized that the denatured protein concentration was too low, so I Amicon concentrated them, and I realized that I gunked up the bottom of the Amicon tube with something that is probably my protein?

So umm...to all the experts out there:

1) is my inclusion body consistency normal?

2) is the inclusion body supposed to turn brown when exposed to 6 M G-HCl

3) i shouldn't have Amicon concentrated them....that screws it up...right?

Thanks in advance!