2 replies to this topic

[Ainara]

Hi,
I have been trying to get protein from trizol. The SDS 1% does not disolve the protein and I tryied urea (10M) and DTT (50mM),(It disolves everything). The problem is that the western blot does not go. Has anybody succeed in western blots from trizol proteins? If so, what do you do?

[sean]

what do u mean the western doesnt go?

i have never used trizol so i cant comment on that... but try precipitating ur proteins after 10M Urea( how did u get it so concentrated anyway? most ppl use 6-8M and have hard time gettin it to dissolve...)

that should remove excess amt of urea .
but to begin with urea should affect the sds page...  

if u have used guHcl then sds ppt's out in u will get rid of Gu Hcl using the same method( TCA ppt)

gd luck

[eCORI]

If I understand your question correctly, you used TriZol to extract proteins? I used TriZol before, and afaik it's used to extract total RNA. TriZol is phenol + guanidinium isothocyanate. After addition of TriZol to tissue/cells, and subsequent centrifugation, proteins and DNA will remain in organic phase while RNA goes to aqueous phase.

Can you please tell me what modifications you made to standard TriZol protocol to allow its use in protein extraction? Thanks very much.

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(edited to say: sorry, i checked the trizol literature again and found the protein extraction protocol at the back. thanks)

(Edited by eCORI at 6:44 pm on April 15, 2001)