We want to detect phosphorylated proteins using the ProQ Diamond (life technologies) and Sypro Ruby (Bio-Rad) stains. PeppermintStick phosphorylated protein standard was used to test this. We stained the PeppermintStick with ProQ Diamons, optimized the image and quantified the fluorescent intensities. Then did the same with Sypro Ruby. According to the insert, calculation of the ProQ diamond (D) / Sypro Ruby (S) ratio is then a measure of the phosphorylation of a protein.
The D/S ratio of the phosphorylated proteins was about 0.05. That of the unphosphorylated proteins was about 0.003. According to the product insert, you would expect a D/S ratio of about 8.0 for phosphorylated proteins (instead of our 0.05) and 0.05 for unphosphorylated proteins (instead of our 0.003).
Did we do something wrong?
Alexander, it might be useful to upload a couple of images if you can but the first thing that comes to mind is that you may have over-destained after staining with Pro-Q. It can be pretty easy to lose the stain from phospho-protein bands. Typically, I will only destain for 2 or 3 minutes, depending how high the background appears by eye.
The other thing to keep in mind is that, in practise, Pro-Q has a pretty narrow linear range (despite claims by the manufacturer). I generally only trust it for 'yes/no'-type experiments rather than quantitative analysis of phosphorylation.
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