Re: Western blot OD analysis

Posted by Wolfgang Schechinger on Apr 17, 2010; 10:04am
URL: http://imagej.273.s1.nabble.com/Western-blot-OD-analysis-tp3688571p3688573.html

Dear Youngmee,

please tell us how you obtained the image you want to evaluate (X-ray film, fluorescence scanner, etc etc)

Wo

-------- Original-Nachricht --------
> Datum: Fri, 16 Apr 2010 19:02:04 -0400
> Von: Youngmee Sul <[hidden email]>
> An: [hidden email]
> Betreff: Western blot OD analysis

> 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. ctrl + 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?

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