quantifications of western blots with imagej

#QUANTIFICATIONS OF WESTERN BLOTS WITH IMAGEJ HOW TO#
#QUANTIFICATIONS OF WESTERN BLOTS WITH IMAGEJ MANUAL#

With appropriate calculations, sample signals can be accurately normalized to housekeeping proteins designated as internal loading controls to obtain quantitative western blot data. It is important to avoid signal saturation in the normalization controls as well as the target protein, or accurate quantitation will not be possible. The relative intensity becomes nonlinear in shape and levels off, indicating virtually the same intensity for higher and higher amounts of protein. They include β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and α-tubulin, which have been shown to become saturated at common lysate loading amounts (e.g., 30–50 μg/well). Figure 1 shows some common normalization controls known as housekeeping proteins, or HKPs. When a chemiluminescent signal becomes saturated, the ability to relate signal intensity to protein abundance is lost. When transitioning your western blotting experiments from qualitative to quantitative analysis, the most important attribute to address is signal saturation. Choosing the correct normalization method for your quantitative western blot is critical for obtaining reliable and reproducible results. Normalization corrects for unavoidable errors that occur during the western blot process, including sample loading or effects from electrophoresis, transfer, or sample concentration. If that's the case, then that may make a huge difference, especially when considering that this plating time was not even mentioned in the paper.Normalization is required to accurately assess differences in target abundance. After talking to my collaborator, we saw that the difference may have been in the amount of time the cells were plated prior to treatment (2 days for the posted images, 1 day for the experiment that didn't work).

#QUANTIFICATIONS OF WESTERN BLOTS WITH IMAGEJ MANUAL#

I was exploring this mainly because I was not seeing a difference in my second experiment (which I did not post here) when I looked at the 'autocontrast' image, but I did see 2-fold when I did manual contrast. That might also tell me that when I do manual contrast, I might be accentuating differences to magnitudes that may not be true.

#QUANTIFICATIONS OF WESTERN BLOTS WITH IMAGEJ HOW TO#

I'm trying to see how to set ImageJ to do quantifications on a 16-bit scale. Even the TIFF image, which is 16-bit, is being quantified with 8-bit settings. The quantifications are being done on an 8-bit scale, which is going to saturate at around 23000. This is the one on which I reduced the background over the entire image, as carried out on the imager itself. This is the original, 'autocorrected' version that the imager spits out. The TIFF is the original provided by the imager, with image exposure of 6 minutes. Does anyone have a good read on whether what I'm doing is acceptable, or should I just do my quantification on the original, non-autocorrected TIFF?ĮDIT: I am including an example of what I'm talking about. Comparing the ImageJ quantification between the finished picture and the original TIFF (which, BTW, is not the same as the original autocorrected image) is giving me nightmares. However, I keep wondering whether this might be considered image manipulation. I was justifying this as merely doing 'shorter' exposures, albeit in reverse. This, I have been finding myself adjusting the 'contrast' (if that is what it is rather than black intensity), which brings out the contrasts between bands better and reduces the saturation of the highly intense bands (the bands would remain the same intensity according to ImageJ, whereas everything else is reduced). However, I notice that the autocorrect function gives a lot of background and band saturation, and this is usually seen even at lower exposure times (semi-auto frequently tells me I have to expose for greater than 10 minutes, but I know that is almost never true). Our lab has started working with the Amersham AI600 imager, which has given some very sensitive blots, thus reducing the amount of antibody we use.

Quantifications of western blots with imagej