Hi,
I am currently working with S. cerevisiae.
general rule of thumbs for yeast cell lysis is to use 0.2M NaOH incubation followed by lysis using high urea buffer (containing 100mM DTT) at 70oC in my lab.
recently I realized addition or removal of DTT can actually change the pattern of my western blot so dramatic and one without adding DTT gives me the logical outcomes while others don’t.
With that I am wondering if the presence of DTT in the lysis buffer actually helps cell lysis procedures or just interfere with protein oxidation and di-sulfide bond formations. Also I wonder if anyone actually happen to come across similar issues.
Many thanks in advance.
Cheers
Don’t know what is logical outcome of your experiment. DTT is a chemical which reacts with the protein it has nothing to do with lysis, people use it to reduce disulfide ; sometimes helps in columns ( like in GST tagged protein purification DTT helps). Never used S. cerevisiae but never heard anyone using 100 mM DTT
Chang Seok Lee Generally DTT do not affect the cell lysis in yeast and filamentous fungi. However, the concentration you have mentioned is too high, we usually use 1mM DTT in lysis buffer, as protease inhibitor.
However, in your case if your protein contains disulphide bond then DTT would be a problem. DTT reduces the di-sulphide bonds, which in some cases causes irreparable loss of structure and activity to your protein.
If you want to get rid of DTT at any step during purification after the lysis, dialyse your sample, and check if you still get the logical outcome.
Best wishes
Thanks all for kind answers!
Generally with lysis using bead beater or sheroplasting, i used to use 1 mM DTT.
But with direct lysis using HU buffer, protocol suggests using 100mM DTT. So there is a big confussion in myself as to why 100mM DTT is even necessary... but it seems DTT addition (either none or 100mM) does affect my western blot.
So is there any possibility that it would actually affect antibody sensitivity or whatnots?
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