[Protein-analysis] "No i pojechalimy" [So, we go now!] - Juri Gagarin after his start on Wostock 1

Julio Cortazar juliocortazar at o2.pl
Tue Aug 15 07:26:27 EST 2006


Hi DK,

Today I made first step in my exp. Telling the truth I'm little bit
worried. When I was adding (drop by drop, over 30min for 4mL)
solubilized proteins of my bacteria (in 6M GuHCl 100mM phosphate
buffer, 100mM NaCl and 20mM betamercaptoethanol, centrifuged 20min
12500g to pellet insoluble deribs) to the refolding buffers (have 3 of
them combination of pH & redox system GSSH:GSH) the buffers turned from
clear/without colour to cloudy one (I'mean looking like diluted milk).

Isn't it aggregation?

Of course I realize two things: one is that inclusion bodies
solubilized using GuHCl contains also different proteins than my,
recombinant one and they can aggregate even in refolding buffer, even
better I will pellet them before loading on the Ni-NTA column), second
thing: not all of my protein will renature correctly, maybe 1%, maybe
10%, don't know. Nevertheless is it natural, this kind of cloudy
turbidity in refolding buffer?

Now it is stirred in a cold room with Naazide and PMSF. I plan to spin
it after 24h. Who knows what will be in supernatant?:)

Julio



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