3 replies to this topic

[vered]

hello
i find a lot of answers in this forum which are really helpful if someone has experience with IP from nuclear extract i would love to read.
the buffers to get the nuclear extract usually have a high salt concentration or denaturing components which might abolish the interaction with my protein when ill try to IP. any knowledge of how this can be handled?
thank you
V

[laurequillo]

vered, on Jun 22 2010, 11:43 PM, said:

hello
i find a lot of answers in this forum which are really helpful if someone has experience with IP from nuclear extract i would love to read.
the buffers to get the nuclear extract usually have a high salt concentration or denaturing components which might abolish the interaction with my protein when ill try to IP. any knowledge of how this can be handled?
thank you
V

Actually I had no problem just using the same buffers I use for nuclear extraction. but if you want you just adjust the final volume with the same buffer without salt, and add the antibody.(which denaturing components do you use in yor buffers?)

[vered]

laurequillo, on Jun 22 2010, 08:56 PM, said:

vered, on Jun 22 2010, 11:43 PM, said:

hello
i find a lot of answers in this forum which are really helpful if someone has experience with IP from nuclear extract i would love to read.
the buffers to get the nuclear extract usually have a high salt concentration or denaturing components which might abolish the interaction with my protein when ill try to IP. any knowledge of how this can be handled?
thank you
V

Actually I had no problem just using the same buffers I use for nuclear extraction. but if you want you just adjust the final volume with the same buffer without salt, and add the antibody.(which denaturing components do you use in yor buffers?)

hello
and thanks for your reply
in the high salt buffers i dont use any denaturing components because i want to keep the integrity of the proteins. if i dont do IP i use DTT or SDS. what kind of buffer do you use for your nuclear extract?
thanks you
v

[laurequillo]

vered, on Jun 23 2010, 01:54 PM, said:

laurequillo, on Jun 22 2010, 08:56 PM, said:

vered, on Jun 22 2010, 11:43 PM, said:

hello
i find a lot of answers in this forum which are really helpful if someone has experience with IP from nuclear extract i would love to read.
the buffers to get the nuclear extract usually have a high salt concentration or denaturing components which might abolish the interaction with my protein when ill try to IP. any knowledge of how this can be handled?
thank you
V

Actually I had no problem just using the same buffers I use for nuclear extraction. but if you want you just adjust the final volume with the same buffer without salt, and add the antibody.(which denaturing components do you use in yor buffers?)

hello
and thanks for your reply
in the high salt buffers i dont use any denaturing components because i want to keep the integrity of the proteins. if i dont do IP i use DTT or SDS. what kind of buffer do you use for your nuclear extract?
thanks you
v

So, if you think your buffer has too much salt, just dilute it x times and do the Ip.

My buffer is:

20mM Hepes pH 7.9
400mM NaCl
1mM EDTA
1mM EGTA
1mM BetaMercaptoethanol
Protease Inhibitors

And I have no problem pulling down my protein

Edited by laurequillo, 23 June 2010 - 04:24 AM.