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Query regarding the use of ImageJ software for the western Blot Analysis

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Shivang Parikh

Query regarding the use of ImageJ software for the western Blot Analysis

Dear Madame/Sir,

Greetings of the day!!!

I am a PhD student at The Hebrew University. I am using the ImageJ software
for the quantification analysis of the western blots by following
way:---Normally I invert the image and select the area of interest (band)
and then measure the mean values keeping the area for all the lanes
constant. Then I select the background from the each lane and subtract it
from the mean value of respective lanes.

But i am not fully satisfied with this approach so i found another method
with the below link for quantification of the blot...

http://howtowesternblot.net/data-analysis-3/quantification/

But still I have some query in my mind and want to ask that which is the
appropriate protocol to quantify the western blots. As mentioned in the
above link or the way i am doing, as in the above link it doesn't take
background into consideration. If in case we don't keep the area constant
then how can we further proceed with the analysis.

Thanking you in anticipation for your favorable reply. I hope to hear from
you.

Regards
*Shivang Parikh*

*U BE GREEN, LEAVE IT ON SCREEN*

------------------------------------------------------------------------------------------------------------------------------

The Hebrew University of Jerusalem, Faculty of Medicine
Department of Biochemistry and Molecular Biology,
Lab Room No. 237,
Second Floor - The Octav and Marcela Botnar Medical Research Building,
PO Box 12272 Jerusalem 91120,
ISRAEL
Cell: - +972-548076106
E-Mail:- [hidden email]

--------------------------------------------------------------------------------------------------------------------------------

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Joel Sheffield

Re: Query regarding the use of ImageJ software for the western Blot Analysis

Hi Shivang,

There is a detailed description of how to use the gel scanning algorithm in
the manual: http://rsbweb.nih.gov/ij/docs/guide/146-30.html . Scroll down
to section 30.13. You will see that if you scan the whole track, you can
get a reasonable approximation of the local background, and eliminate it
from your calculation. Of course, if your gel is saturated, you'll get
saturated data (although sometimes the width of the band is proportional to
the concentration, but that's a different story)

The important aspect of any of this analysis is to set up standards of
concentration so that you can make sense of the results. Especially with
Westerns, there is a general problem of non-linearity.

Joel Sheffield

Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL: http://astro.temple.edu/~jbs

On Wed, May 14, 2014 at 12:32 PM, Shivang Parikh
<[hidden email]>wrote:

> Dear Madame/Sir,
>
> Greetings of the day!!!
>
> I am a PhD student at The Hebrew University. I am using the ImageJ software
> for the quantification analysis of the western blots by following
> way:---Normally I invert the image and select the area of interest (band)
> and then measure the mean values keeping the area for all the lanes
> constant. Then I select the background from the each lane and subtract it
> from the mean value of respective lanes.
>
> But i am not fully satisfied with this approach so i found another method
> with the below link for quantification of the blot...
>
> http://howtowesternblot.net/data-analysis-3/quantification/
>
> But still I have some query in my mind and want to ask that which is the
> appropriate protocol to quantify the western blots. As mentioned in the
> above link or the way i am doing, as in the above link it doesn't take
> background into consideration. If in case we don't keep the area constant
> then how can we further proceed with the analysis.
>
> Thanking you in anticipation for your favorable reply. I hope to hear from
> you.
>
>
> Regards
> *Shivang Parikh*
>
>
> *U BE GREEN, LEAVE IT ON SCREEN*
>
>
> ------------------------------------------------------------------------------------------------------------------------------
>
>
> The Hebrew University of Jerusalem, Faculty of Medicine
> Department of Biochemistry and Molecular Biology,
> Lab Room No. 237,
> Second Floor - The Octav and Marcela Botnar Medical Research Building,
> PO Box 12272 Jerusalem 91120,
> ISRAEL
> Cell: - +972-548076106
> E-Mail:- [hidden email]
>
>
> --------------------------------------------------------------------------------------------------------------------------------
>
>
>
>
> ------------------------------------------------------------------------------------------------------------------------------
> This message contains confidential and/or privileged information meant only
> for the addressee. If you are not the addressee or authorized to receive
> this message on behalf of the addressee, you must not use, copy, disclose
> or take any action based on this message or any information herein. If you
> have received this message in error, please destroy this message along with
> attachments and advise the sender immediately by reply e-mail. Thank you
> for your co-operation.
>
> ------------------------------------------------------------------------------------------------------
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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gankaku

Re: Query regarding the use of ImageJ software for the western Blot Analysis

In reply to this post by Shivang Parikh

Hi Shivang,

Besides the fact that Western Blot "quantification" in the context of
densiometry is at its best semi-quantitative. It has many pitfalls which
make it kind of not-so-reliable (my opinion).
In the case you can visually detect a difference between bands (in
comparison to the loading control) I would potentially believe a certain
stated difference (potentially starting at values around 20-25%).

Radio-Immuno-Assays are potentially the most reliable way of quantification
in this context. Thereafter fluorescently labeled blots and then methods
like ECL etc.
The latter method has the problem that enzyme reaction and substrate
turnover are most likely not a linear process (as, in addition, is the
darkening of x-ray films if those are used)

The following publications might help you to get insight into pitfalls and
instructions of semi-quantitative evaluation of Western Blots:

http://www.ncbi.nlm.nih.gov/pubmed/19517440
http://www.ncbi.nlm.nih.gov/pubmed/?term=Improved+semiquantitative+Western+blot+technique+with+increased+quantification+range

Hope this helps.

Kind regards,
Jan

2014-05-14 18:32 GMT+02:00 Shivang Parikh <[hidden email]>:

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CEO: Dr. rer. nat. Jan Brocher
phone: +49 (0)6234 917 03 39
mobile: +49 (0)176 705 746 81
e-mail: [hidden email]
info: [hidden email]
inquiries: [hidden email]
web: www.biovoxxel.de

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Shivang Parikh

Re: Query regarding the use of ImageJ software for the western Blot Analysis

In reply to this post by Joel Sheffield

Dear Professor Sheffield and Professor Jan Brocher,

Thank you very much for your respective candid replies. But still I have some queries. I load 60 ug protein in each lane of the western blot and it's not a purified protein it's a crude protein. So I get the whole lysate of bands when I check it after the transfer with Ponseau. Now what I wanted to ask you is when a get the band of my interest (which includes from control and up-regulated or down-regulated conditions) there is difference in the clear visible increase and decrease in the bands; i.e. Control is less intense and the band area is also less compared to the up-regulated band area which shows a diffuse type of pattern more intense up and gets less intense downwards, but still it's a whole band. So how am I supposed to select which area of the band. I know that the area of the measurement window in the software should be constant for every lane, and shouldn't change.

But I have following queries:-

Query 1:-whether Am I suppose to take the band area with the maximum area into consideration? If yes then the up-regulated bands show more intensity at the top and less at the bottom. Considering the whole area will the imageJ take average of it? Will it show decrease in the actual result compared to only specific area of the band where the intensity is maximum?

Query 2:- whether the area of selection window for measuring the bands be kept constant ?

Query 3:- if I only consider the more intense area only in compare to the whole area in the up-regulated protein condition then it gives more mean values compare to that of the whole band area (intense + diffused) ?

Please guide me that what is the correct method to take which areas of bands into consideration for the quantification to determine the fold increase decrease ?

Thanking you in anticipation.

Sincerely
Shivang

Sent from my iPad

> On 14 May 2014, at 23:15, "JOEL B. SHEFFIELD" <[hidden email]> wrote:
>
> Hi Shivang,
>
> There is a detailed description of how to use the gel scanning algorithm in
> the manual: http://rsbweb.nih.gov/ij/docs/guide/146-30.html . Scroll down
> to section 30.13. You will see that if you scan the whole track, you can
> get a reasonable approximation of the local background, and eliminate it
> from your calculation. Of course, if your gel is saturated, you'll get
> saturated data (although sometimes the width of the band is proportional to
> the concentration, but that's a different story)
>
> The important aspect of any of this analysis is to set up standards of
> concentration so that you can make sense of the results. Especially with
> Westerns, there is a general problem of non-linearity.
>
> Joel Sheffield
>
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
> e-mail: [hidden email]
> URL: http://astro.temple.edu/~jbs
>
>
> On Wed, May 14, 2014 at 12:32 PM, Shivang Parikh
> <[hidden email]>wrote:
>
>> Dear Madame/Sir,
>>
>> Greetings of the day!!!
>>
>> I am a PhD student at The Hebrew University. I am using the ImageJ software
>> for the quantification analysis of the western blots by following
>> way:---Normally I invert the image and select the area of interest (band)
>> and then measure the mean values keeping the area for all the lanes
>> constant. Then I select the background from the each lane and subtract it
>> from the mean value of respective lanes.
>>
>> But i am not fully satisfied with this approach so i found another method
>> with the below link for quantification of the blot...
>>
>> http://howtowesternblot.net/data-analysis-3/quantification/
>>
>> But still I have some query in my mind and want to ask that which is the
>> appropriate protocol to quantify the western blots. As mentioned in the
>> above link or the way i am doing, as in the above link it doesn't take
>> background into consideration. If in case we don't keep the area constant
>> then how can we further proceed with the analysis.
>>
>> Thanking you in anticipation for your favorable reply. I hope to hear from
>> you.
>>
>>
>> Regards
>> *Shivang Parikh*
>>
>>
>> *U BE GREEN, LEAVE IT ON SCREEN*
>>
>>
>> ------------------------------------------------------------------------------------------------------------------------------
>>
>>
>> The Hebrew University of Jerusalem, Faculty of Medicine
>> Department of Biochemistry and Molecular Biology,
>> Lab Room No. 237,
>> Second Floor - The Octav and Marcela Botnar Medical Research Building,
>> PO Box 12272 Jerusalem 91120,
>> ISRAEL
>> Cell: - +972-548076106
>> E-Mail:- [hidden email]
>>
>>
>> --------------------------------------------------------------------------------------------------------------------------------
>>
>>
>>
>>
>> ------------------------------------------------------------------------------------------------------------------------------
>> This message contains confidential and/or privileged information meant only
>> for the addressee. If you are not the addressee or authorized to receive
>> this message on behalf of the addressee, you must not use, copy, disclose
>> or take any action based on this message or any information herein. If you
>> have received this message in error, please destroy this message along with
>> attachments and advise the sender immediately by reply e-mail. Thank you
>> for your co-operation.
>>
>> ------------------------------------------------------------------------------------------------------
>>
>> --
>> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

Joel Sheffield

Re: Query regarding the use of ImageJ software for the western Blot Analysis

JOEL B. SHEFFIELD <[hidden email]>
8:57 PM (8 minutes ago)

to ImageJ
Hi Shivang,

It might help us if you were to make available some of the images that you
are working with. You might post them to some neutral site, or, possibly
attach them to an e-mail.

At any rate, as both I and Professor Brocher have indicated, bands on a gel
are complex structures that form as a result of electrophoretic force
acting on charged samples, moving them through a physically resistive
medium. At the same time as the bands migrate, they diffuse, as a function
of concentration effects. Thus, the proper measurement of a band of
material should include the integrated density of the entire band, with the
background subtracted. As you realize, in many gels, the precise value for
the background is often variable and difficult to determine. Many
commercial companies have invested significant effort into devising ways
around these challenges. The ImageJ solutions are an approximation that
generally works quite well, if your needs for precision are not that great,
and you don't have to analyze closely migrating bands. However, it is
absolutely necessary for you to develop your own standardization by
creating gels with known amounts of protein. If you do this, you will be
able to determine for yourself whether peak height alone is a sufficient
measurement in your system.

I have prepared a laboratory exercise that I use in my undergraduate class
in cell biology. Perhaps it will be of some help. Unfortunately, the IJ
listserv rejects pdf files, so I have sent it to you privately. I mention
it here, in case others are interested. Although it is essentially what is
presented in the ImageJ manual referenced above, I'd be happy to share.

Best of Luck,

Joel

Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL: http://astro.temple.edu/~jbs

On Fri, May 16, 2014 at 5:22 PM, Shivang Parikh <[hidden email]>wrote:

> Dear Professor Sheffield and Professor Jan Brocher,
>
> Thank you very much for your respective candid replies. But still I have
> some queries. I load 60 ug protein in each lane of the western blot and
> it's not a purified protein it's a crude protein. So I get the whole lysate
> of bands when I check it after the transfer with Ponseau. Now what I wanted
> to ask you is when a get the band of my interest (which includes from
> control and up-regulated or down-regulated conditions) there is difference
> in the clear visible increase and decrease in the bands; i.e. Control is
> less intense and the band area is also less compared to the up-regulated
> band area which shows a diffuse type of pattern more intense up and gets
> less intense downwards, but still it's a whole band. So how am I supposed
> to select which area of the band. I know that the area of the measurement
> window in the software should be constant for every lane, and shouldn't
> change.
>
> But I have following queries:-
>
> Query 1:-whether Am I suppose to take the band area with the maximum area
> into consideration? If yes then the up-regulated bands show more intensity
> at the top and less at the bottom. Considering the whole area will the
> imageJ take average of it? Will it show decrease in the actual result
> compared to only specific area of the band where the intensity is maximum?
>
> Query 2:- whether the area of selection window for measuring the bands be
> kept constant ?
>
> Query 3:- if I only consider the more intense area only in compare to the
> whole area in the up-regulated protein condition then it gives more mean
> values compare to that of the whole band area (intense + diffused) ?
>
> Please guide me that what is the correct method to take which areas of
> bands into consideration for the quantification to determine the fold
> increase decrease ?
>
> Thanking you in anticipation.
>
> Sincerely
> Shivang
>
> Sent from my iPad
>
> > On 14 May 2014, at 23:15, "JOEL B. SHEFFIELD" <[hidden email]> wrote:
> >
> > Hi Shivang,
> >
> > There is a detailed description of how to use the gel scanning algorithm
> in
> > the manual: http://rsbweb.nih.gov/ij/docs/guide/146-30.html . Scroll
> down
> > to section 30.13. You will see that if you scan the whole track, you can
> > get a reasonable approximation of the local background, and eliminate it
> > from your calculation. Of course, if your gel is saturated, you'll get
> > saturated data (although sometimes the width of the band is proportional
> to
> > the concentration, but that's a different story)
> >
> > The important aspect of any of this analysis is to set up standards of
> > concentration so that you can make sense of the results. Especially with
> > Westerns, there is a general problem of non-linearity.
> >
> > Joel Sheffield
> >
> >
> >
> > Joel B. Sheffield, Ph.D
> > Department of Biology
> > Temple University
> > Philadelphia, PA 19122
> > Voice: 215 204 8839
> > e-mail: [hidden email]
> > URL: http://astro.temple.edu/~jbs
> >
> >
> > On Wed, May 14, 2014 at 12:32 PM, Shivang Parikh
> > <[hidden email]>wrote:
> >
> >> Dear Madame/Sir,
> >>
> >> Greetings of the day!!!
> >>
> >> I am a PhD student at The Hebrew University. I am using the ImageJ
> software
> >> for the quantification analysis of the western blots by following
> >> way:---Normally I invert the image and select the area of interest
> (band)
> >> and then measure the mean values keeping the area for all the lanes
> >> constant. Then I select the background from the each lane and subtract
> it
> >> from the mean value of respective lanes.
> >>
> >> But i am not fully satisfied with this approach so i found another
> method
> >> with the below link for quantification of the blot...
> >>
> >> http://howtowesternblot.net/data-analysis-3/quantification/
> >>
> >> But still I have some query in my mind and want to ask that which is the
> >> appropriate protocol to quantify the western blots. As mentioned in the
> >> above link or the way i am doing, as in the above link it doesn't take
> >> background into consideration. If in case we don't keep the area
> constant
> >> then how can we further proceed with the analysis.
> >>
> >> Thanking you in anticipation for your favorable reply. I hope to hear
> from
> >> you.
> >>
> >>
> >> Regards
> >> *Shivang Parikh*
> >>
> >>
> >> *U BE GREEN, LEAVE IT ON SCREEN*
> >>
> >>
> >>
> ------------------------------------------------------------------------------------------------------------------------------
> >>
> >>
> >> The Hebrew University of Jerusalem, Faculty of Medicine
> >> Department of Biochemistry and Molecular Biology,
> >> Lab Room No. 237,
> >> Second Floor - The Octav and Marcela Botnar Medical Research Building,
> >> PO Box 12272 Jerusalem 91120,
> >> ISRAEL
> >> Cell: - +972-548076106
> >> E-Mail:- [hidden email]
> >>
> >>
> >>
> --------------------------------------------------------------------------------------------------------------------------------
> >>
> >>
> >>
> >>
> >>
> ------------------------------------------------------------------------------------------------------------------------------
> >> This message contains confidential and/or privileged information meant
> only
> >> for the addressee. If you are not the addressee or authorized to receive
> >> this message on behalf of the addressee, you must not use, copy,
> disclose
> >> or take any action based on this message or any information herein. If
> you
> >> have received this message in error, please destroy this message along
> with
> >> attachments and advise the sender immediately by reply e-mail. Thank you
> >> for your co-operation.
> >>
> >>
> ------------------------------------------------------------------------------------------------------
> >>
> >> --
> >> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
> >
> > --
> > ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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