z*******a
发帖数: 175 | 1 my comment on comments of Nemo and Biogene (see below). by the way, I like
these two names.
1. Once upon a time, I did not take any <0.03 % input as a real binding when
I used antisera not purified IgG.
But I saw some papers in top journals have data published like 0.00something
%input, really confusing. do
you two or 达人 have any comments?
2. I would say enrichment folds instead of fold enrichment. I should
calculate folds relative to normal serum
control when antisera are used or IgG control when purified IgG antibodies
are used, which means every
single sample must have a normal serum or IgG control, which makes me sick.
So I often times use non
related DNA region around a housekeeping gene promoter as reference. I do
NOT think it is correct. but I
have never seen a single reviewer for not bad journals has problem with it,
which makes me even sicker. So I
always want to avoid ChIP whenever I can.
拙见， 见笑了 |
m******5
发帖数: 1383 | 2 Thank you much for your great contribution to my rookie question^^
I think non-related region negative control is convincing theoretically, if
not taking non- specific DNA precipitation into account: different DNA
region might have different tendencies of non specific precipitation (I also
learned that this issue can be simply avoid by using dynal beads)
A parable phenomenon is that when I was doing invitro protein interaction
assay such like GST pull-down and co-ip, the reviewer did not question a
pulldown or co ip assay without GST or IgG control when I have binding
motif mapped out by truncation.
Thank you again!
when
00something
【在 z*******a 的大作中提到】
: my comment on comments of Nemo and Biogene (see below). by the way, I like
: these two names.
: 1. Once upon a time, I did not take any <0.03 % input as a real binding when
: I used antisera not purified IgG.
: But I saw some papers in top journals have data published like 0.00something
: %input, really confusing. do
: you two or 达人 have any comments?
: 2. I would say enrichment folds instead of fold enrichment. I should
: calculate folds relative to normal serum
: control when antisera are used or IgG control when purified IgG antibodies
|
a****o
发帖数: 1786 | 3 fold of enrichment is a better measurement than IP efficiency. fold greater
than 4 is a good number.
Low IP efficiency sometimes come from that YFP binds to certain target only
in a fraction of cells.
when
00something
【在 z*******a 的大作中提到】
: my comment on comments of Nemo and Biogene (see below). by the way, I like
: these two names.
: 1. Once upon a time, I did not take any <0.03 % input as a real binding when
: I used antisera not purified IgG.
: But I saw some papers in top journals have data published like 0.00something
: %input, really confusing. do
: you two or 达人 have any comments?
: 2. I would say enrichment folds instead of fold enrichment. I should
: calculate folds relative to normal serum
: control when antisera are used or IgG control when purified IgG antibodies
|
z*******a
发帖数: 175 | 4 I used solid tissue of animals. You are talking about binding of
exogenously transfected protein to
endogenous regulatory elements. I am wondering how you do transfection
efficiency control in the first place
if it is transient transfection. equal exogenously expressed protein makes
your data comparable between
treatments and repeats. I do NOT have confidence to get evenly transfected
efficiency all the time.
greater
only
【在 a****o 的大作中提到】
: fold of enrichment is a better measurement than IP efficiency. fold greater
: than 4 is a good number.
: Low IP efficiency sometimes come from that YFP binds to certain target only
: in a fraction of cells.
:
: when
: 00something
|
m******5
发帖数: 1383 | 5 I have another question: I didn't see much people have immune-precipitated
protein shown in their paper(even for those who did overexpression protein
immuneprecipitation) ,which is very very weird, because it should not be
very hard theoretically. without immune-precipitation efficiency, by which
they can convince the reviewer or themselves that the binding of DNA is
reasonable?
【在 z*******a 的大作中提到】
: I used solid tissue of animals. You are talking about binding of
: exogenously transfected protein to
: endogenous regulatory elements. I am wondering how you do transfection
: efficiency control in the first place
: if it is transient transfection. equal exogenously expressed protein makes
: your data comparable between
: treatments and repeats. I do NOT have confidence to get evenly transfected
: efficiency all the time.
:
: greater
|
z*******a
发帖数: 175 | 6 absolutely right.
The first thing I would do is to run western to see how much protein pulled
down. This is very important to
check if your antibody is working in protein cross-linked condition. But I
think it is reasonable not to have the
data published in journal because of space limit.
【在 m******5 的大作中提到】
: I have another question: I didn't see much people have immune-precipitated
: protein shown in their paper(even for those who did overexpression protein
: immuneprecipitation) ,which is very very weird, because it should not be
: very hard theoretically. without immune-precipitation efficiency, by which
: they can convince the reviewer or themselves that the binding of DNA is
: reasonable?
|
B*****e
发帖数: 1005 | 7 Thank you for your kind reply.
IgG content of Antibody may not be equal to purified IgG,
Why don't calculate fold enrichment relative to KO cell line using same
antibody?
2. I would say enrichment folds instead of fold enrichment. I should
calculate folds relative to normal serum
control when antisera are used or IgG control when purified IgG antibodies
are used, which means every
single sample must have a normal serum or IgG control, which makes me sick.
So I often times use non
related DNA region around a housekeeping gene promoter as reference. I do
NOT think it is correct. but I
have never seen a single reviewer for not bad journals has problem with it,
which makes me even sicker. So I
always want to avoid ChIP whenever I can.
【在 z*******a 的大作中提到】
: absolutely right.
: The first thing I would do is to run western to see how much protein pulled
: down. This is very important to
: check if your antibody is working in protein cross-linked condition. But I
: think it is reasonable not to have the
: data published in journal because of space limit.
|
z*******a
发帖数: 175 | 8 It is always the best when the KO animals (null mutants) or cell lines are
available |
