3 replies to this topic

[Erik Edstrom]

I'm planning to use the Trizol protocol for protein isolation. Has anyone used this sucessfully for protein analyzed by western blot? The product protocol is a bit ambivalent as to how the protein should be redissolved. Dissolution in 1% SDS is suggested but for spectrophotometric quantitation the same protocol states less than 0.1% SDS should be used. Anyone with experience from trizol in these issues?

[Ainara]

I have been trying this for a few months, and my protein was not recognized by the antibody. Also, SDS-electrophoresis migration looked different than the protein extracted with conventional methods. The only way I could disolve the protein from Trizol was with urea.
I suggest you not to use trizol for western blots.
Good luck!

[AVondracek]

I have used Trizol sucessfully in an organ localization Western. I had to heat the protein in 1% SDS for about 20 min at >50C, before the proteins would resuspend enough for Bradford. The SDS obviously interferes with Bradford, but you can still see you protein if you run SDS negative control. The protein did act a bit different then sera alone, but protein extraction by Trizol is possible.

[jnilsen]

I have also successfully used Trizol for protein extraction and subsequent Western. Heat the sample in 1% SDS ~50oC to redissolve. We used BCA to measure protein concentration diluting the sample 1:10, thus the resulting 0.1% SDS didn't affect readings.