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

Posted by gankaku on May 15, 2014; 7:06am
URL: http://imagej.273.s1.nabble.com/Query-regarding-the-use-of-ImageJ-software-for-the-western-Blot-Analysis-tp5007737p5007745.html

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]>:

> 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*
>
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