On Jan 13, 6:50 am, "Iraz Toprak Aydin" <iraz.ay...@epfl.ch> wrote: > Hello everyone! > > I will soon try to isolate proteins from melanocytes, I need to get both > Triton X-100 soluble and insoluble fractions. For this purpose I will make > > this lysis buffer; 50 mM Tris, 150 mM NaCl, 1% Triton X-100, 10 mM > EDTA, pH 7.2. And for the insoluble fraction I will use the same buffer with > 8M urea. So my questions are: > > 1. Can I autoclave the lysis buffer? Or should I filter it? (22um or > 45um) >
The product information sheet for TX-100 that Sigma distributes claims it it stable to autoclaving, but honestly, I don't know why you would want to. I don't think I've ever felt the need to autoclave a Triton- containing solution. Exposure to light is supposed to promote peroxide formation from Triton, so you might want to store it in the dark. When I typically make a solution like that, usually I just make it by diluting clean stock solutions that I usually have on hand. > 2. For the lysis buffer with urea, can I also prepare this one before > and store it? Or shall I add the urea before use? > > 3. Can I prepare a urea stock solution? Which concentration and storage > conditions would you recommend? > Urea can decompose on storage, especially when warmed, to isocyanates and ammonia, which can modify proteins, so it's generally better not to let it sit around awhile. If you feel the urge to autoclave something, what I would do is make up a 5x or 10x concentrated stock of the salts and buffers and autoclave that. Keep some solution of 10% Triton aliquoted and stored cold (maybe even with nitrogen on top if you're feeling particularly compulsive). Then on the day of the experiment just dilute the stocks into fresh water, either with or without the amount of urea you need. For example, let's say you need 50 ml of urea extraction buffer. Put 24 g of urea in a 50 ml conical tube, add 5 ml of 10x buffer, 5 ml of 10% Triton (which will be a lot easier to pipet than 0.5 ml of 100% Triton), and enough water to bring to 50 ml, shake until dissolved. But that's just me. Everybody has his own way of doing things. Nick -- Nick Theodorakis nick_theodora...@hotmail.com contact form: http://theodorakis.net/contact.html _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
