how to normalize ELISA signal??# Biology - 生物学
P*b
1 楼
求教，是选被引用最多的， 还是 IF最高的，还是最对口的啊？
IF的高的和比较对口都是这两年才发的，引用率还没上去。被引用最多的文章是当年刚
开始读博士的第一篇文章。文章一般，就是年头有了，引用率比较高。
IF的高的和比较对口都是这两年才发的，引用率还没上去。被引用最多的文章是当年刚
开始读博士的第一篇文章。文章一般，就是年头有了，引用率比较高。
w*e
2 楼
做了六次实验，两个sample：实验组＋control组。每次signal都很不一样，
但每次实验组跟control组相比都有同样的区别，请教如何作图？
现在想的一个偷懒的办法是把每次control组的signal都normalize为1，
再对实验组的相对数据做统计分析做error bar，不知是否acceptable？
多谢！
但每次实验组跟control组相比都有同样的区别，请教如何作图？
现在想的一个偷懒的办法是把每次control组的signal都normalize为1，
再对实验组的相对数据做统计分析做error bar，不知是否acceptable？
多谢！
p*c
3 楼
你有篇Science，另外有篇BBRC最对口，还有篇JBC引用率最高
你说选哪篇s？
【在 P****b 的大作中提到】
: 求教，是选被引用最多的， 还是 IF最高的，还是最对口的啊？
: IF的高的和比较对口都是这两年才发的，引用率还没上去。被引用最多的文章是当年刚
: 开始读博士的第一篇文章。文章一般，就是年头有了，引用率比较高。
你说选哪篇s？
【在 P****b 的大作中提到】
: 求教，是选被引用最多的， 还是 IF最高的，还是最对口的啊？
: IF的高的和比较对口都是这两年才发的，引用率还没上去。被引用最多的文章是当年刚
: 开始读博士的第一篇文章。文章一般，就是年头有了，引用率比较高。
I*y
4 楼
use free probe as control
【在 w******e 的大作中提到】
: 做了六次实验，两个sample：实验组＋control组。每次signal都很不一样，
: 但每次实验组跟control组相比都有同样的区别，请教如何作图？
: 现在想的一个偷懒的办法是把每次control组的signal都normalize为1，
: 再对实验组的相对数据做统计分析做error bar，不知是否acceptable？
: 多谢！
【在 w******e 的大作中提到】
: 做了六次实验，两个sample：实验组＋control组。每次signal都很不一样，
: 但每次实验组跟control组相比都有同样的区别，请教如何作图？
: 现在想的一个偷懒的办法是把每次control组的signal都normalize为1，
: 再对实验组的相对数据做统计分析做error bar，不知是否acceptable？
: 多谢！
M*n
5 楼
do you make a stardard curve each time for your analyte?
you must make standards every time when you run the assay, or you cannot compare all the data together.
you must make standards every time when you run the assay, or you cannot compare all the data together.
w*e
6 楼
the thing is, I'm not measuring the value of the target sample,
instead, I have a fix protein conc. for my target sample, 0 protein
for my control. My purpose is instead to demonstrate that my assay
indeed worked in detecting the protein. hence the headache...
maybe I worried too much. all I need to show is that the assay
worked, anyway.
compare all the data together.
【在 M*****n 的大作中提到】
: do you make a stardard curve each time for your analyte?
: you must make standards every time when you run the assay, or you cannot compare all the data together.
instead, I have a fix protein conc. for my target sample, 0 protein
for my control. My purpose is instead to demonstrate that my assay
indeed worked in detecting the protein. hence the headache...
maybe I worried too much. all I need to show is that the assay
worked, anyway.
compare all the data together.
【在 M*****n 的大作中提到】
: do you make a stardard curve each time for your analyte?
: you must make standards every time when you run the assay, or you cannot compare all the data together.
M*n
7 楼
to demonstrate your assay is working,
you need use an alternative method (say conventional sanwich ELISA) to
measure your protein to make the standard curve and make a correlation to what you get with your method.
otherwise, you can not claim your method is working.
Besides, what I see is your method may have too much variation and no good
retestability. that is bad. you need improve that.
【在 w******e 的大作中提到】
: the thing is, I'm not measuring the value of the target sample,
: instead, I have a fix protein conc. for my target sample, 0 protein
: for my control. My purpose is instead to demonstrate that my assay
: indeed worked in detecting the protein. hence the headache...
: maybe I worried too much. all I need to show is that the assay
: worked, anyway.
:
: compare all the data together.
you need use an alternative method (say conventional sanwich ELISA) to
measure your protein to make the standard curve and make a correlation to what you get with your method.
otherwise, you can not claim your method is working.
Besides, what I see is your method may have too much variation and no good
retestability. that is bad. you need improve that.
【在 w******e 的大作中提到】
: the thing is, I'm not measuring the value of the target sample,
: instead, I have a fix protein conc. for my target sample, 0 protein
: for my control. My purpose is instead to demonstrate that my assay
: indeed worked in detecting the protein. hence the headache...
: maybe I worried too much. all I need to show is that the assay
: worked, anyway.
:
: compare all the data together.
w*e
8 楼
what you get with your method.
This is a good point. I'm too lazy to do that as it's a proof-of-concept
experiment, but I do appreciate the input:)
Just curious, wouldn't ELISA signal always jump around (in terms of absolute
signal value)? It happens in my hand all the time even with Ab ELISA-_-
【在 M*****n 的大作中提到】
: to demonstrate your assay is working,
: you need use an alternative method (say conventional sanwich ELISA) to
: measure your protein to make the standard curve and make a correlation to what you get with your method.
: otherwise, you can not claim your method is working.
: Besides, what I see is your method may have too much variation and no good
: retestability. that is bad. you need improve that.
l*a
9 楼
我听着挺靠谱啊，为啥不行呢？btw，我不是搞纯生物的，意见不具代表性。
【在 w******e 的大作中提到】
: 做了六次实验，两个sample：实验组＋control组。每次signal都很不一样，
: 但每次实验组跟control组相比都有同样的区别，请教如何作图？
: 现在想的一个偷懒的办法是把每次control组的signal都normalize为1，
: 再对实验组的相对数据做统计分析做error bar，不知是否acceptable？
: 多谢！
【在 w******e 的大作中提到】
: 做了六次实验，两个sample：实验组＋control组。每次signal都很不一样，
: 但每次实验组跟control组相比都有同样的区别，请教如何作图？
: 现在想的一个偷懒的办法是把每次control组的signal都normalize为1，
: 再对实验组的相对数据做统计分析做error bar，不知是否acceptable？
: 多谢！
M*n
10 楼
ELISA is quite sensitive and hence big variations, but the retest
reliabiligy is an important issue you need address. My boss always bugs me
with this, whenever I want to run a new assay.
have you calculated Z' score?
I would suggest you run at least triplicates in parallele and calculate sd
for each sample and Z'for the series of your samples and controls. your Z'
must be > 0.5 the higher the better.
BTW, get a set of new pippettes.
absolute
【在 w******e 的大作中提到】
:
: what you get with your method.
: This is a good point. I'm too lazy to do that as it's a proof-of-concept
: experiment, but I do appreciate the input:)
: Just curious, wouldn't ELISA signal always jump around (in terms of absolute
: signal value)? It happens in my hand all the time even with Ab ELISA-_-
reliabiligy is an important issue you need address. My boss always bugs me
with this, whenever I want to run a new assay.
have you calculated Z' score?
I would suggest you run at least triplicates in parallele and calculate sd
for each sample and Z'for the series of your samples and controls. your Z'
must be > 0.5 the higher the better.
BTW, get a set of new pippettes.
absolute
【在 w******e 的大作中提到】
:
: what you get with your method.
: This is a good point. I'm too lazy to do that as it's a proof-of-concept
: experiment, but I do appreciate the input:)
: Just curious, wouldn't ELISA signal always jump around (in terms of absolute
: signal value)? It happens in my hand all the time even with Ab ELISA-_-
w*e
11 楼
I'm pretty dumb when it comes to statistics, so you need to take it slow:)
so by retest reliability you mean duplicate in the same experiment?
I actually got those data from totally independent exps. I did duplicate
in one exp and they look similar, though I never bother to check how
similar-_-
I never used Z score b4-_- wiki ing...
in fact the point of my current project is the creation of an affinity
agent pair that "should" be useful in sandwich assay. I just want to have
a quick and dirty assay to stress the point, and save room for a
follow-up paper with nice sensor set-up and STD curve and all. Hopefully
my reviewer won't be too strict on this hehe
now you are being mean:) you admit the variation yourself man:)
【在 M*****n 的大作中提到】
: ELISA is quite sensitive and hence big variations, but the retest
: reliabiligy is an important issue you need address. My boss always bugs me
: with this, whenever I want to run a new assay.
: have you calculated Z' score?
: I would suggest you run at least triplicates in parallele and calculate sd
: for each sample and Z'for the series of your samples and controls. your Z'
: must be > 0.5 the higher the better.
: BTW, get a set of new pippettes.
:
: absolute
so by retest reliability you mean duplicate in the same experiment?
I actually got those data from totally independent exps. I did duplicate
in one exp and they look similar, though I never bother to check how
similar-_-
I never used Z score b4-_- wiki ing...
in fact the point of my current project is the creation of an affinity
agent pair that "should" be useful in sandwich assay. I just want to have
a quick and dirty assay to stress the point, and save room for a
follow-up paper with nice sensor set-up and STD curve and all. Hopefully
my reviewer won't be too strict on this hehe
now you are being mean:) you admit the variation yourself man:)
【在 M*****n 的大作中提到】
: ELISA is quite sensitive and hence big variations, but the retest
: reliabiligy is an important issue you need address. My boss always bugs me
: with this, whenever I want to run a new assay.
: have you calculated Z' score?
: I would suggest you run at least triplicates in parallele and calculate sd
: for each sample and Z'for the series of your samples and controls. your Z'
: must be > 0.5 the higher the better.
: BTW, get a set of new pippettes.
:
: absolute
M*n
12 楼
i re-read your previous post, i think in your case the Z' will be low.
normalizing the control to 1 is OK, but it is not a clean way to do it. I
think you need an independent ELISA as a standard curve. do you always do a
dosage curve?
【在 w******e 的大作中提到】
: I'm pretty dumb when it comes to statistics, so you need to take it slow:)
: so by retest reliability you mean duplicate in the same experiment?
: I actually got those data from totally independent exps. I did duplicate
: in one exp and they look similar, though I never bother to check how
: similar-_-
: I never used Z score b4-_- wiki ing...
: in fact the point of my current project is the creation of an affinity
: agent pair that "should" be useful in sandwich assay. I just want to have
: a quick and dirty assay to stress the point, and save room for a
: follow-up paper with nice sensor set-up and STD curve and all. Hopefully
normalizing the control to 1 is OK, but it is not a clean way to do it. I
think you need an independent ELISA as a standard curve. do you always do a
dosage curve?
【在 w******e 的大作中提到】
: I'm pretty dumb when it comes to statistics, so you need to take it slow:)
: so by retest reliability you mean duplicate in the same experiment?
: I actually got those data from totally independent exps. I did duplicate
: in one exp and they look similar, though I never bother to check how
: similar-_-
: I never used Z score b4-_- wiki ing...
: in fact the point of my current project is the creation of an affinity
: agent pair that "should" be useful in sandwich assay. I just want to have
: a quick and dirty assay to stress the point, and save room for a
: follow-up paper with nice sensor set-up and STD curve and all. Hopefully
w*e
13 楼
I only did one dosage curve -- it has the right trend, but I don't intend
to include the data anyway. the point of my project is, here I generated
a new aptamer pair for sensor ppl to play with (just google thrombin
sensor you'll know how those ppl are lol~), for assurance I demonstrated
they can give higher signal with target than w/o, and the signal is
specific to the target. I'll leave claims about sensitivity & stuff to
sensor ppl. end of story (hopefully).
anyway, thx for your time~
a
【在 M*****n 的大作中提到】
: i re-read your previous post, i think in your case the Z' will be low.
: normalizing the control to 1 is OK, but it is not a clean way to do it. I
: think you need an independent ELISA as a standard curve. do you always do a
: dosage curve?
to include the data anyway. the point of my project is, here I generated
a new aptamer pair for sensor ppl to play with (just google thrombin
sensor you'll know how those ppl are lol~), for assurance I demonstrated
they can give higher signal with target than w/o, and the signal is
specific to the target. I'll leave claims about sensitivity & stuff to
sensor ppl. end of story (hopefully).
anyway, thx for your time~
a
【在 M*****n 的大作中提到】
: i re-read your previous post, i think in your case the Z' will be low.
: normalizing the control to 1 is OK, but it is not a clean way to do it. I
: think you need an independent ELISA as a standard curve. do you always do a
: dosage curve?
M*n
14 楼
how do you couple this to a sensor?
I am very intersted in this since I am also working on developing high-
throughput quatitative measurement.
is this for a grant? if so, don't worry too much about.
if it is for a quick publication, maybe you want to make it a bit better
looking.
【在 w******e 的大作中提到】
: I only did one dosage curve -- it has the right trend, but I don't intend
: to include the data anyway. the point of my project is, here I generated
: a new aptamer pair for sensor ppl to play with (just google thrombin
: sensor you'll know how those ppl are lol~), for assurance I demonstrated
: they can give higher signal with target than w/o, and the signal is
: specific to the target. I'll leave claims about sensitivity & stuff to
: sensor ppl. end of story (hopefully).
: anyway, thx for your time~
:
: a
I am very intersted in this since I am also working on developing high-
throughput quatitative measurement.
is this for a grant? if so, don't worry too much about.
if it is for a quick publication, maybe you want to make it a bit better
looking.
【在 w******e 的大作中提到】
: I only did one dosage curve -- it has the right trend, but I don't intend
: to include the data anyway. the point of my project is, here I generated
: a new aptamer pair for sensor ppl to play with (just google thrombin
: sensor you'll know how those ppl are lol~), for assurance I demonstrated
: they can give higher signal with target than w/o, and the signal is
: specific to the target. I'll leave claims about sensitivity & stuff to
: sensor ppl. end of story (hopefully).
: anyway, thx for your time~
:
: a
w*e
15 楼
it's very easy to couple aptamers to sensor. in fact sensor field
is dominated by aptamer as affinity agent as the stability &
reusability is a big deal for sensors.
back to your questions, you can do thiol-gold, EDC-NHS, biotin-SA...
all you need is to synthesize the aptamer w/ desired functional group.
so are you doing arrays? or beads, like luminex? you may want to check
out larry gold's recent publication
I'd certainly like to, if it doesn't take too much time. any suggestions?
I'm confident the performance could be improved though, as I just
made up some condition and started collecting data. The whole business
of optimizing aptamer sensor can get tricky. Again,
just search thrombin aptamers hehe
【在 M*****n 的大作中提到】
: how do you couple this to a sensor?
: I am very intersted in this since I am also working on developing high-
: throughput quatitative measurement.
: is this for a grant? if so, don't worry too much about.
: if it is for a quick publication, maybe you want to make it a bit better
: looking.
is dominated by aptamer as affinity agent as the stability &
reusability is a big deal for sensors.
back to your questions, you can do thiol-gold, EDC-NHS, biotin-SA...
all you need is to synthesize the aptamer w/ desired functional group.
so are you doing arrays? or beads, like luminex? you may want to check
out larry gold's recent publication
I'd certainly like to, if it doesn't take too much time. any suggestions?
I'm confident the performance could be improved though, as I just
made up some condition and started collecting data. The whole business
of optimizing aptamer sensor can get tricky. Again,
just search thrombin aptamers hehe
【在 M*****n 的大作中提到】
: how do you couple this to a sensor?
: I am very intersted in this since I am also working on developing high-
: throughput quatitative measurement.
: is this for a grant? if so, don't worry too much about.
: if it is for a quick publication, maybe you want to make it a bit better
: looking.
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