Biotinilated proteins. Problems with Hens buffer - (Aug/24/2006 )
Hi!
I am trying to detect nitrosylated proteins through the biotin-switch method. I have problem to prepare the Hens buffer (250 mM HEPES pH 7.7, 1mM EDTA, 0.1mM Neocuproine, 1%SDS). If I prepare the HEPES and adjust the pH I cannot disolve the rest and if I disolve all and finally I adjust the pH when it reach around 7.0 something start to precipitate.
Thanks very much in advance for the help.
-spanishbusybee-
QUOTE (spanishbusybee @ Aug 24 2006, 12:21 PM)
Hi!
I am trying to detect nitrosylated proteins through the biotin-switch method. I have problem to prepare the Hens buffer (250 mM HEPES pH 7.7, 1mM EDTA, 0.1mM Neocuproine, 1%SDS). If I prepare the HEPES and adjust the pH I cannot disolve the rest and if I disolve all and finally I adjust the pH when it reach around 7.0 something start to precipitate.
Thanks very much in advance for the help.
I am trying to detect nitrosylated proteins through the biotin-switch method. I have problem to prepare the Hens buffer (250 mM HEPES pH 7.7, 1mM EDTA, 0.1mM Neocuproine, 1%SDS). If I prepare the HEPES and adjust the pH I cannot disolve the rest and if I disolve all and finally I adjust the pH when it reach around 7.0 something start to precipitate.
Thanks very much in advance for the help.
I don't know what's the problem with your buffer preparation, but if you success to detect nitrosylated proteins by this method, let me know
I would like to apply it on some of my samples, but one of my colleagues told me that only one person in the world (in a extremely specialized lab in USA) seem to be able to do it! So, all I could do with this method would be unsuccessful.
If one day you say to me you succeeded, I'll try it ;-)
-behappy736-
QUOTE (behappy736 @ Sep 16 2006, 12:19 PM)
QUOTE (spanishbusybee @ Aug 24 2006, 12:21 PM)
Hi!
I am trying to detect nitrosylated proteins through the biotin-switch method. I have problem to prepare the Hens buffer (250 mM HEPES pH 7.7, 1mM EDTA, 0.1mM Neocuproine, 1%SDS). If I prepare the HEPES and adjust the pH I cannot disolve the rest and if I disolve all and finally I adjust the pH when it reach around 7.0 something start to precipitate.
Thanks very much in advance for the help.
I don't know what's the problem with your buffer preparation, but if you success to detect nitrosylated proteins by this method, let me know
I would like to apply it on some of my samples, but one of my colleagues told me that only one person in the world (in a extremely specialized lab in USA) seem to be able to do it! So, all I could do with this method would be unsuccessful.
If one day you say to me you succeeded, I'll try it ;-)
Dont worry my desperate friend. I were desperate once, so you are not alone. Your buffer seems to have too much neocuproine (10X higher), so it may be precipitating. Better use 0.01mM. See modifications by janssen-heininger group (Proc Natl Acad Sci U S A. 2004 June 15; 101(24): 8945–8950). Her work is reliable and rigorous. BTW in Netherlands. Godspeed!
-Robertzar-