L*******e
发帖数: 2153 | 1 I am planning to prepare coIP samples and use mass spec to get a protein
binding profile. By comparing control and mutant groups, I am hoping to
find targets that are more disease relevant. I use magnetic beads for Co-IP.
Now here comes the questions:
Would it be good enough to just use SDS buffer for elution? I know this is
the case if my next step is western blotting.
If not, would an acidic glycine buffer followed by a basic buffer (Tris-HCl
) be sufficient since this only results elution of native protein complexes?
What are the other methods you recommend for removing the SDS from protein
samples in addition to running them on a gel? Columns?
Thank you!!!! |
L*******e
发帖数: 2153 | 2 anyone ......?
IP.
HCl
complexes?
【在 L*******e 的大作中提到】
: I am planning to prepare coIP samples and use mass spec to get a protein
: binding profile. By comparing control and mutant groups, I am hoping to
: find targets that are more disease relevant. I use magnetic beads for Co-IP.
: Now here comes the questions:
: Would it be good enough to just use SDS buffer for elution? I know this is
: the case if my next step is western blotting.
: If not, would an acidic glycine buffer followed by a basic buffer (Tris-HCl
: ) be sufficient since this only results elution of native protein complexes?
: What are the other methods you recommend for removing the SDS from protein
: samples in addition to running them on a gel? Columns?
|
o******p
发帖数: 55 | 3
IP.
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Samples can be separated on a gel and subsequently cleaned up to remove SDS.
HCl
complexes?
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Changing the pH works, but usually denatures proteins. I used to do elutions
with ammonium hydroxide (pH>10). It is both efficient and convenient. It
can be removed simply by speed vac.
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Filter assisted sample preparation (FASP) is a popular choice.
【在 L*******e 的大作中提到】
: I am planning to prepare coIP samples and use mass spec to get a protein
: binding profile. By comparing control and mutant groups, I am hoping to
: find targets that are more disease relevant. I use magnetic beads for Co-IP.
: Now here comes the questions:
: Would it be good enough to just use SDS buffer for elution? I know this is
: the case if my next step is western blotting.
: If not, would an acidic glycine buffer followed by a basic buffer (Tris-HCl
: ) be sufficient since this only results elution of native protein complexes?
: What are the other methods you recommend for removing the SDS from protein
: samples in addition to running them on a gel? Columns?
|
L*******e
发帖数: 2153 | 4 OMG~~~~~~~~~
LOVE IT!
Thanks!!!!!
SDS.
elutions
【在 o******p 的大作中提到】
:
: IP.
: ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
: Samples can be separated on a gel and subsequently cleaned up to remove SDS.
: HCl
: complexes?
: ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
: Changing the pH works, but usually denatures proteins. I used to do elutions
: with ammonium hydroxide (pH>10). It is both efficient and convenient. It
: can be removed simply by speed vac.
|
K******S
发帖数: 10109 | 5 LOL, orbitrap
SDS.
elutions
【在 o******p 的大作中提到】
:
: IP.
: ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
: Samples can be separated on a gel and subsequently cleaned up to remove SDS.
: HCl
: complexes?
: ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
: Changing the pH works, but usually denatures proteins. I used to do elutions
: with ammonium hydroxide (pH>10). It is both efficient and convenient. It
: can be removed simply by speed vac.
|
L*******e
发帖数: 2153 | 6 lol
what was that ......profile pic?
【在 K******S 的大作中提到】
: LOL, orbitrap
:
: SDS.
: elutions
|
x**2
发帖数: 255 | 7 Orbitrap是一种mass spectrometer
你的样品可以用Orbitrap分析，也可以用其它的mass spectrometer
KingofMS回帖的意思是：三楼回你帖的人用户名叫Orbitrap，真好玩儿
话说你可以多和KingofMS聊聊，他对mass spectrometer的各种应用非常了解 |
x**2
发帖数: 255 | 8 lol是
laughing out loud
的简写 |
L*******e
发帖数: 2153 | 9 谢谢，您的指点！
【在 x**2 的大作中提到】
: Orbitrap是一种mass spectrometer
: 你的样品可以用Orbitrap分析，也可以用其它的mass spectrometer
: KingofMS回帖的意思是：三楼回你帖的人用户名叫Orbitrap，真好玩儿
: 话说你可以多和KingofMS聊聊，他对mass spectrometer的各种应用非常了解
|
z********1
发帖数: 4 | 10 没做过MASS SPEC,但据说一般coIP时用双tag比较好，用peptide置换洗脱，这样特异性
好，比较容易成功。 |
S*********s
发帖数: 304 | 11 1.FLAG-HA double tag->stable cell line
2.Flag elute/HA elute
3.send out for MS sequencing
IP.
HCl
complexes?
【在 L*******e 的大作中提到】
: I am planning to prepare coIP samples and use mass spec to get a protein
: binding profile. By comparing control and mutant groups, I am hoping to
: find targets that are more disease relevant. I use magnetic beads for Co-IP.
: Now here comes the questions:
: Would it be good enough to just use SDS buffer for elution? I know this is
: the case if my next step is western blotting.
: If not, would an acidic glycine buffer followed by a basic buffer (Tris-HCl
: ) be sufficient since this only results elution of native protein complexes?
: What are the other methods you recommend for removing the SDS from protein
: samples in addition to running them on a gel? Columns?
|
