I am looking at protocols for measuring proton pump activity of V-ATPase. Most of the papers use Acridine Orange to staining purified lysosomes. Then after adding ATP, measure the quenching of fluorescence. But no one really describe the excitation and emission wavelength as AO has multiple pairs for different purpose. Could any expert help me to specify it? Another question is about the principle of this assay. Does it mean more acidity of lysosomes would quench AO fluorescence? Thanks a million in advance. Looking forward to your valuable answers.
Yes, it is pH based. v-ATPase pump protons to make the lumen acidic. Use v-ATPase inhibitor bafilomycin A1 to examine that v-type ATPases are doing the trick.
Ex 490 nm and Em 530 nm as per the following ref
http://link.springer.com/article/10.1007/BF01941777
I haven't worked with quenching per se.
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