Trition X100 - (Nov/06/2006 )
Can any body have an experience of working with Trition X100. I used it and it settles at the bottom if i keep my lysis solution then it does not mix at all and again settles at the bottom.
So if it settle at the bottom and it will not work well. Can any body have an experience of working with it and any good tip.
regards
What's in your lysis solution?
I've never had any trouble with Triton X100 once it's in solution.
Are you mixing your solution thoroughly when you first make it up?
Triton X100 will froth when agitated but you should thoroughly mix it when you make your lysis solution up and leave it until it settles.
Edit: Edited for clarity.
Unless you use Triton X114 which doesn't froth at all
I have NP-10 Lysis buffer. which is 50 mM NaH2PO4 and 300 mM NaCl and 10mM Imidazole.
First I use lysozyme then after half an hr i add detergent. you shake the lysis solution or not. Trition x 100 is seem ok at room temperature but Trition X114 ok at ice.
maybe you could prepare a 10x triton X100, (let's say 10% if you lyse with 1%)
you have to be patient because it's difficult to solubilize, you can put it at 37°C and vortex well.
(do not prepare just before need ! you need let's say 1 hour to solubilze)
then you just have to add 1/10 of the 10X triton to your sample. easy to pipet and already solubilized. 
I have to prepare 10x in my NP10 buffer. Because i am directly mixing the detergent from company supplied detergent into my lysis solution mean first suspend bacterial pellet into NP-10 buffer then 0.2 mg lysozyme then wait for 45 minutes then add detergents 1 percent.
You mean i will dilute detergent into buffer and then use its 1/10 conc into my lysis solution.
Second i am fear about my protein degradation at 37 C but i will try.
regards
you have to be patient because it's difficult to solubilize, you can put it at 37°C and vortex well.
(do not prepare just before need ! you need let's say 1 hour to solubilze)
then you just have to add 1/10 of the 10X triton to your sample. easy to pipet and already solubilized.
I make a solution of STET which has NaCl, Tris, EDTA and Triton X-100. To get the triton into solution, I make it up in a graduated cylinder and spend the next hour or so gently inverting the cylinder to get it into solution. It does take a while and you definately don't want to shake it as tons of bubbles form. However, once this goes into solution, I have no problem keeping it there.
Is it possible to make up your lysis buffer in a similar manner? I'm not sure if you are adding the Triton directly to an individual reaction, but if you can make up a batch of the lysis buffer I think it would help. Good luck!
Triton will froth when agitated but you should thoroughly mix it when you make your lysis solution up and leave it until it settles.
Unless you use Triton X114 which doesn't froth at all
Well, maybe, I don't know because I have no experience with Triton X114. But then, weren't we talking about Triton X100? *winky*
You mean i will dilute detergent into buffer and then use its 1/10 conc into my lysis solution.
Second i am fear about my protein degradation at 37 C but i will try.
regards
maybe you could prepare a 10x triton X100, (let's say 10% if you lyse with 1%)
you have to be patient because it's difficult to solubilize, you can put it at 37°C and vortex well.
(do not prepare just before need ! you need let's say 1 hour to solubilze)
then you just have to add 1/10 of the 10X triton to your sample. easy to pipet and already solubilized.
you don't need to put your sample at 37°C
you put your 10x triton X-100 at 37°C, when it's solubilized, you can put it at 4°C.
then you put your bacteria pellet in NP-10 buffer, then you add your lysozyme, and then you add the triton X-100 10% (you can solubilize it in NP-10 buffer), at 4°C if you want (it's already solubilized)