Green/red dual fluorescence detection of total protein and alkaline phosphate-conjugated probes on blotting membranes

Abstract

A two-color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY FL-X to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl(DDAO)-phosphate in combination with alkaline-phosphatase-conjugated reporter molecules. This results in all proteins in the profile being visualized as green signal while those detected specifically with the alkaline-phosphatase conjugate appear as red signal. The dichromatic detection system is broadly compatible with a wide range of analytical imaging devices including UV epi- or transilluminators combined with photographic or charge-coupled device (CCD) cameras, xenon-arc sources equipped with appropriate excitation/emission filters, and dual laser gel scanners outfitted with a 473 nm second-harmonic generation or 488 nm argon-ion laser as well as a 633 nm helium-neon or 635 nm diode laser. The dichromatic detection method permits detection of low nanogram amounts of protein and allows for unambiguous identification of target proteins relative to the entire protein profile on a single electroblot, obviating the need to run replicate gels that would otherwise require visualization of total proteins by silver staining and subsequent alignment with chemiluminescent or colorimetric signals generated on electroblots.

Development of BODIPY FL VH032 as a High-Affinity and Selective von Hippel–Lindau E3 Ligase Fluorescent Probe and Its Application in a Time-Resolved Fluorescence Resonance Energy-Transfer Assay

“…When excited at 340 nm, terbium emits light centered at 490 nm with a bandwidth of ca. 10 nm which overlaps well with the excitation spectrum of BODIPY FL (with a peak at 504 nm and a relatively broad bandwidth) . The acceptor fluorophore BODIPY FL is then excited and emits light at 520 nm.…”

“…10 nm which overlaps well with the excitation spectrum of BODIPY FL (with a peak at 504 nm and a relatively broad bandwidth). 29 The acceptor fluorophore BODIPY FL is then excited and emits light at 520 nm. The 520 nm emission signal is due to the TR-FRET.…”

3′-Terminal Repair-Powered Dendritic Nanoassembly of Polyadenine Molecular Beacons for One-Step Quantification of Alkaline Phosphatase in Human Serum

“…1 The ALP normal level of adults is 40−190 U/L in human serum, 2 and the abnormal level may induce a variety of human diseases (e.g., bone diseases, cardiovascular diseases, and inflammatory disorders) 3−5 and cancers (e.g., oral, prostate, and liver cancers). 6−8 Importantly, ALP is widely used as the labeling tracer in biomolecules monitoring, 9 histochemical staining 10 and gene expression analysis 11 due to its broad substrate specificity, high catalytic activity, good stability, and low cost. Therefore, simple, specific, and sensitive detection of the ALP activity is essential to biomedical research, cancer therapy, and clinical diagnoses.…”

“…Alkaline phosphatase (ALP), an essential monophosphate hydrolase existing in all human tissues, can catalyze the hydrolysis of phosphate groups in carbohydrates, proteins, and nucleic acids, playing important roles in regulating multiple biological processes (e.g., cellular division, proliferation, apoptosis, and signal transduction) . The ALP normal level of adults is 40–190 U/L in human serum, and the abnormal level may induce a variety of human diseases (e.g., bone diseases, cardiovascular diseases, and inflammatory disorders) − and cancers (e.g., oral, prostate, and liver cancers). − Importantly, ALP is widely used as the labeling tracer in biomolecules monitoring, histochemical staining and gene expression analysis due to its broad substrate specificity, high catalytic activity, good stability, and low cost. Therefore, simple, specific, and sensitive detection of the ALP activity is essential to biomedical research, cancer therapy, and clinical diagnoses.…”