Quantification problem: more values despite weaker band?
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Hey,
I´m having problems quantifying some of my blots. The problem is as follows: I have 2 lanes on my blot - one lane showing a thick protein band, the second one showing a much much fainter band. Surprisingly, I always get a higher RawIntDen for the clearly weaker band... I really have no idea what the problem might be. After development, I save my pictures as .tif (16 bit) and change it to 8bit in ImageJ. Furthermore I convert the image so that black bands appear white and the background is black. Could it be problem that I save the pics as 16bit at the beginning? I would be very grateful for some helping hints! Thanks! Verena |
Re: Quantification problem: more values despite weaker band?
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Hi Verena,
You may have the lut reversed. If you move the cursor over the bands in the image, do you get a high or a low value for the "darker" band? It seems that you are using the Analyze particles routine for the analysis. You might want to try the Gel Analyzer. 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, Apr 25, 2014 at 10:33 AM, Rena2014 <[hidden email]>wrote: > Hey, > > I´m having problems quantifying some of my blots. The problem is as > follows: > I have 2 lanes on my blot - one lane showing a thick protein band, the > second one showing a much much fainter band. Surprisingly, I always get a > higher RawIntDen for the clearly weaker band... I really have no idea what > the problem might be. > After development, I save my pictures as .tif (16 bit) and change it to > 8bit > in ImageJ. Furthermore I convert the image so that black bands appear white > and the background is black. > Could it be problem that I save the pics as 16bit at the beginning? > I would be very grateful for some helping hints! Thanks! > > Verena > > > > -- > View this message in context: > http://imagej.1557.x6.nabble.com/Quantification-problem-more-values-despite-weaker-band-tp5007430.html > Sent from the ImageJ mailing list archive at Nabble.com. > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html > -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
Re: Quantification problem: more values despite weaker band?
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In reply to this post by Rena2014
Even if the bands appear white they could still have a lower intensity that
the black background. This happens if you inverted the display colors but not really changed the actual intensity values; the image window may say "Inverting LUT' on the info bar. First try applying Image > Lookup Tables > Grays to see the "true" intensities (high intensity as white, low as black). To confirm the intensity values, wave the mouse over a band and over background areas whilst checking the intensity values reported in the ImageJ window. If the bands are lower intensity then you have to invert your image again, or don't invert it in the first place. Good luck! -Esteban On Fri, Apr 25, 2014 at 7:33 AM, Rena2014 <[hidden email]>wrote: > Hey, > > I´m having problems quantifying some of my blots. The problem is as > follows: > I have 2 lanes on my blot - one lane showing a thick protein band, the > second one showing a much much fainter band. Surprisingly, I always get a > higher RawIntDen for the clearly weaker band... I really have no idea what > the problem might be. > After development, I save my pictures as .tif (16 bit) and change it to > 8bit > in ImageJ. Furthermore I convert the image so that black bands appear white > and the background is black. > Could it be problem that I save the pics as 16bit at the beginning? > I would be very grateful for some helping hints! Thanks! > > Verena > > > > -- > View this message in context: > http://imagej.1557.x6.nabble.com/Quantification-problem-more-values-despite-weaker-band-tp5007430.html > Sent from the ImageJ mailing list archive at Nabble.com. > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html > -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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Hi,
Thanks already for your comments on my quantification problem! So far I followed a protocol from the University of Missouri (http://support.dalton.missouri.edu/index.php/wiki/Public:Quantifying_Color_Intensity/). As recommended, I checked the "true intensities" and in some of my images I see high values for the black bands and low values for the white background (if the image is not inverted). So, clearly here is something inverted, right? |
Re: Quantification problem: more values despite weaker band?
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Hi Rena,
This protocol has some serious errors in it. However, the most glaring is that the scanning is done on a "conventional flatbed scanner". You might be aware that the light in a scanner will actually pass through your intermediate bands twice--once as it leaves the light source, and again when it is reflected from the back side of the scanner. As a result, your readings might not be particularly quantitative. Some scanners have specific modes for transmission. It's much better to use one of those, or even a carefully done photograph. In all cases, I would suggest that you carry out a true calibration of bands to ensure that you are getting linear results. 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 Mon, Apr 28, 2014 at 9:40 AM, Rena2014 <[hidden email]>wrote: > Hi, > > Thanks already for your comments on my quantification problem! > So far I followed a protocol from the University of Missouri > ( > http://support.dalton.missouri.edu/index.php/wiki/Public:Quantifying_Color_Intensity/ > ). > As recommended, I checked the "true intensities" and in some of my images I > see high values for the black bands and low values for the white background > (if the image is not inverted). So, clearly here is something inverted, > right? > > > > -- > View this message in context: > http://imagej.1557.x6.nabble.com/Quantification-problem-more-values-despite-weaker-band-tp5007430p5007470.html > Sent from the ImageJ mailing list archive at Nabble.com. > > -- > ImageJ mailing list: http://imagej.nih.gov/ij/list.html > -- ImageJ mailing list: http://imagej.nih.gov/ij/list.html |
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Dear Joel, Fortunately, we use an LAS imaging system from Fuji to develop our gels, though there is no need for an extra scanning. But nevertheless, one should definitely be more careful andbetter think about protocols before just using them... Thanks anyway! Verena 2014-04-28 19:45 GMT+02:00 JOEL B. SHEFFIELD [via ImageJ] <[hidden email]>: Hi Rena, -- Verena Ihrig
Institut für Physiologie MA 2/48 Universitätsstraße 150 44801 Bochum 0234/ 32-29411 |
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