Re: Western blot OD analysis
Posted by
Jos=?ISO-8859-1?Q?=C3=A9?= Gomes on
Apr 17, 2010; 12:05am
URL: http://imagej.273.s1.nabble.com/Western-blot-OD-analysis-tp3688571p3688576.html
hello.
i used the image to wb analisys too. however I select all bands in the same
moment in the horizontal line. not each band. because if you select each
band the number genereated is one, if you select all bands in the same time
the number genereated is tottaly different from results obtained from each
band separated. i already observed these differences in my analisys.
i believe that when you select all bands in the same time the program read
the bands and produced a result based in their interactions and the results
are more coeherent. try it to you see if your results correspond to that
observed vissually.
good look
Gomes/Brazil
2010/4/16 Youngmee Sul <
[hidden email]>
> I performed western blot of transfected cos7 cells to determine the levels
> of our protein of interest. This is our first time using imagej to
> quantitatively analyze western blot. To test imagej's quantitative
> capabilities, we loaded 3ul, 6ul, 12ul and 18ul of the same protein in
> different wells. The gel showed a gradual increase in intensity of the band
> proportionate to the increase in protein loaded.
>
> I want to determine the densitometry of each of these bands. I used the
> following procedure:
> 1. subtract the background with rolling ball radius of 25.
> 2. outline 1st band (usually start with the brightest band) with box
> selection tool
> 3. ctrl + 1
> 4. outline next band
> 5. ctrle + 2
> 6. repeat steps 4 and 5 until all the bands are selected
> 7. ctrl + 3 (the plots appear)
> 8. using the straight line tool, I close the bases of all the inverted
> parabolas.
> 9. using magic wand, select inside the enclosed parabolas to obtain the
> areas
> 10. i used the areas as a measurement of relative intensity.
>
> This method did not yield the appropriate ratios that indicate the correct
> increase in protein loaded.
>
> What am I doing wrong? Will the areas determined from the gel analysis not
> reflect the increase in protein loaded? Is there another method to quantify
> western blots?
>
--
Jose Rosa Gomes -
Mestre em Ciências pelo ICB-USP-área Histologia e Embriologia
Doutor em Biologia Buco-Dental pela FOP/UNICAMP -área Histologia e
Embriologia
Professor Assistente D do Curso de Odontologia da Universidade Estadual de
Ponta Grossa -www.uepg.br-
Editor Chefe do periódico Animal Biology Journal
https://www.novapublishers.com/catalog/product_info.php?products_id=8943 -
New York - USA