Do you add Triton-X in your lysis buffer? - (Dec/17/2007 )
My lysis buffer is: 200mM NaCl,25mM Tris-HCl, 5mM imidazole.pH8.0.
so is Triton-X a necessary component?
-SpringGao-
NaCl is for osmolarity, Tris for pH buffer, imidazole is for chelating ions, there is nothing in your buffer for lysis.
If you want to use this buffer to lyse something, yes you should add triton-X100, or NP-40.
-Missele-
Hi friend
as missele mentioned there is nothing that can lyse your cells.
adding Triton-100 (0.1%) nonionic detergent can do cell lysis and also help in solabilization of unsolable proteins
(can add many undesired proteins to the lysate).
I think that u should first lysed by sonication or homogenization after treatment with lysozyme, then try
different detergents with your protein.
-amtash-
If you need to make a lysis buffer,add triton or sds or other detergents.
-scolix-
QUOTE (SpringGao @ Dec 17 2007, 09:35 PM)
My lysis buffer is: 200mM NaCl,25mM Tris-HCl, 5mM imidazole.pH8.0.
so is Triton-X a necessary component?
so is Triton-X a necessary component?
your lysis buffer should work by hypoosmolarity shock in the case of mammalian cells (cell lines); triton-x solubilizes membranes
-The Bearer-
Thank you everyone.
my former buffer is 500mM NaCl,20mM Tris-HCl, 5mM imidazole, pH8.0. My colleague called it binding buffer. I thought it contain too much salt ion, so reduce it to 200mM.
I prepare cell lysates from E. coli by sonication.
I found some of my proteins are insoluble(pellet has while supernatant no by SDS-PAGE), so I think a detergent is necessary.
-SpringGao-
QUOTE (SpringGao @ Dec 21 2007, 02:07 AM)
Thank you everyone.
my former buffer is 500mM NaCl,20mM Tris-HCl, 5mM imidazole, pH8.0. My colleague called it binding buffer. I thought it contain too much salt ion, so reduce it to 200mM.
I prepare cell lysates from E. coli by sonication.
I found some of my proteins are insoluble(pellet has while supernatant no by SDS-PAGE), so I think a detergent is necessary.
my former buffer is 500mM NaCl,20mM Tris-HCl, 5mM imidazole, pH8.0. My colleague called it binding buffer. I thought it contain too much salt ion, so reduce it to 200mM.
I prepare cell lysates from E. coli by sonication.
I found some of my proteins are insoluble(pellet has while supernatant no by SDS-PAGE), so I think a detergent is necessary.
A lysis buffer should contain:
buffer substance to adjust the pH (e.g. Tris, 10-20 mM should be sifficient to buffer the pH cange induced by lysed cells)
salt (e.g. NaCl for osmolarity. This stabilizes some proteins. isotonic is 154 mM NaCl but if yo use hypotonic conditions (20 mM NaCl), cells will break better)
protease inhibitors: very important as due to the lysis of the cells and compartment membranes the lysate contains a mixture of proteins and proteases which would not "see" each other in intact cells. Therefore proteases can start degrading your protein of interest. Use for example benzamidin, PMSF, Pefabloc or commercially available mixtures like Roche´s Complete. Noe: If you want to purify the protein later via an HIS tag, do not use EDTA as protease nhibitor or elsewhere in the buffer. (Also DTT, bME or other reducing agents will distroy the beads if used in too high concentrations).
lysozyme: the crack the cell wall of E.coli, the addition and incubation of the cells with lysozyme (which can be part of the lysis buffer) is helpfull. Additionally hypotonic buffers will supprt the lysis after lysozyme digestion (without lysozyme, hypotonic buffers do not crack E.coli alone)
DNase: supercoiled DNA can increase the viscocity of the lysate, therefore add some DNase to the lysisbuffer. But note: DNase needs cofactors like Mg (for random strain breaks) or Mn (for blunt ended strain breaks) and some Ca. If you have EDTA or EGTA in the buffer, the concentration of these ions will be lowered!
If you sonicate, DNase it not necessary.
Other ingredients like:
Detergents, RNase are not really necessary. Detergents like Tween, Triton, NP40, etc. will increase solubilisation of membranes and lysis, but soncation in general is much more efficient. Also some proteins will be better soluble with detergents. BUT detergents can interfere with a lot of tests afterwards (enzymatic activity is ofter inhibited, protein interactions are reduced, even protein concentration measurements can be altered, etc.). And detergens are very difficult to remove. Even a purification step like HIS tag, Gelfirltation, etc. will not remove the detergents.
So you shold consider very carefully, whether you really need teh Trition for you lysate.
If your protein is insoluble, a detergent might solubilize it, but it will also denature it most probably! Thats still good for SDS-PAGE or Western, but not for functional assays...
Better try refolding methods via Guanidinium chloride or Urea. They also solubilize a protein but you can try to refold it afterwards to achieve an active protein again.
-Dukes-
QUOTE (Dukes @ Dec 21 2007, 01:41 AM)
QUOTE (SpringGao @ Dec 21 2007, 02:07 AM)
Thank you everyone.
my former buffer is 500mM NaCl,20mM Tris-HCl, 5mM imidazole, pH8.0. My colleague called it binding buffer. I thought it contain too much salt ion, so reduce it to 200mM.
I prepare cell lysates from E. coli by sonication.
I found some of my proteins are insoluble(pellet has while supernatant no by SDS-PAGE), so I think a detergent is necessary.
my former buffer is 500mM NaCl,20mM Tris-HCl, 5mM imidazole, pH8.0. My colleague called it binding buffer. I thought it contain too much salt ion, so reduce it to 200mM.
I prepare cell lysates from E. coli by sonication.
I found some of my proteins are insoluble(pellet has while supernatant no by SDS-PAGE), so I think a detergent is necessary.
A lysis buffer should contain:
buffer substance to adjust the pH (e.g. Tris, 10-20 mM should be sifficient to buffer the pH cange induced by lysed cells)
salt (e.g. NaCl for osmolarity. This stabilizes some proteins. isotonic is 154 mM NaCl but if yo use hypotonic conditions (20 mM NaCl), cells will break better)
protease inhibitors: very important as due to the lysis of the cells and compartment membranes the lysate contains a mixture of proteins and proteases which would not "see" each other in intact cells. Therefore proteases can start degrading your protein of interest. Use for example benzamidin, PMSF, Pefabloc or commercially available mixtures like Roche´s Complete. Noe: If you want to purify the protein later via an HIS tag, do not use EDTA as protease nhibitor or elsewhere in the buffer. (Also DTT, bME or other reducing agents will distroy the beads if used in too high concentrations).
lysozyme: the crack the cell wall of E.coli, the addition and incubation of the cells with lysozyme (which can be part of the lysis buffer) is helpfull. Additionally hypotonic buffers will supprt the lysis after lysozyme digestion (without lysozyme, hypotonic buffers do not crack E.coli alone)
DNase: supercoiled DNA can increase the viscocity of the lysate, therefore add some DNase to the lysisbuffer. But note: DNase needs cofactors like Mg (for random strain breaks) or Mn (for blunt ended strain breaks) and some Ca. If you have EDTA or EGTA in the buffer, the concentration of these ions will be lowered!
If you sonicate, DNase it not necessary.
Other ingredients like:
Detergents, RNase are not really necessary. Detergents like Tween, Triton, NP40, etc. will increase solubilisation of membranes and lysis, but soncation in general is much more efficient. Also some proteins will be better soluble with detergents. BUT detergents can interfere with a lot of tests afterwards (enzymatic activity is ofter inhibited, protein interactions are reduced, even protein concentration measurements can be altered, etc.). And detergens are very difficult to remove. Even a purification step like HIS tag, Gelfirltation, etc. will not remove the detergents.
So you shold consider very carefully, whether you really need teh Trition for you lysate.
If your protein is insoluble, a detergent might solubilize it, but it will also denature it most probably! Thats still good for SDS-PAGE or Western, but not for functional assays...
Better try refolding methods via Guanidinium chloride or Urea. They also solubilize a protein but you can try to refold it afterwards to achieve an active protein again.
Thanks for your detailed advice, especially for the remind about detergent.
We donot use lysozyme to lysis cell currently. sonication solo(it's free of charge after all). Basically, I follow the protocol described in this webpage: http://wolfson.huji.ac.il/purification/Tag...sTag_nature.htm , there are some tips about inclusion body.
QUOTE
If most of the protein remains insoluble after extraction, try
a) To change lysis buffer by adding additives as ß-ME, glycerol, detergents or more NaCl.
B) Re-extract pellet with more buffer,
c) Use more lysis buffer during extraction,
d) Perform a more intensive sonication,
e) Incubate with lysozyme before sonication.
f) Try the denaturating protocol
a) To change lysis buffer by adding additives as ß-ME, glycerol, detergents or more NaCl.
B) Re-extract pellet with more buffer,
c) Use more lysis buffer during extraction,
d) Perform a more intensive sonication,
e) Incubate with lysozyme before sonication.
f) Try the denaturating protocol
and I purified proteins for in purpose to analysis phosphor transfer in vitro. Former researchers use triton X-100 at 0.1%, so it must be applicable to add detergent.
further more, I dialyze my elution to discharge imidazole in it, and I think detergent will become rare.
Also, I'd like to ask my boss buy some lysozyme soon, sonication itself may be laggard these days.
-SpringGao-