Sensitive fluorescence detection of polyphosphate in polyacrylamide gels using 4',6-diamidino-2-phenylindol.
PAGE is commonly used to identify and resolve inorganic polyphosphates (polyP). We now report highly sensitive and specific staining methods for polyP in polyacrylamide gels based on the fluorescent dye, 4',6-diamidino-2-phenylindol (DAPI). DAPI bound to polyP in gels fluoresced yellow while DAPI bound to nucleic acids or glycosaminoglycans fluoresced blue. Inclusion of EDTA prevented staining of glycosaminoglycans by DAPI. We also identified conditions under which DAPI that was bound to polyP (but not nucleic acids or other anionic polymers) rapidly photobleached. This allowed us to develop an even more sensitive and specific negative staining method that distinguishes polyP from nucleic acids and glycosaminoglycans. The lower LOD using DAPI negative staining was 4 pmol (0.3 ng) phosphate per band, compared to conventional toluidine blue staining with a lower LOD of 250 pmol per band.
Smith
Stephanie A
SA
Department of Internal Medicine, College of Medicine, University of Illinois at Urbana-Champaign, 506 S. Mathews, Urbana, IL 61801, USA. sasmith6@uiuc.edu
Morrissey
James H
JH
D004492:Edetic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006025:Glycosaminoglycans / Q000737:chemistry; D007211:Indoles / Q000737:chemistry*; D009696:Nucleic Acids / Q000737:chemistry; D011122:Polyphosphates / Q000032:analysis* / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling / Q000379:methods*
D005456:Fluorescent Dyes; D006025:Glycosaminoglycans; D007211:Indoles; D009696:Nucleic Acids; D011122:Polyphosphates; C007293:DAPI; D004492:Edetic Acid