Biomedical subjects

C C Bean

Publications and source records attributed to C C Bean.

2 recordsLinked to original sources

PubMed1993

Evaluation of nonisotopic labeling and detection techniques for nucleic acid hybridization.

We have used a double-stranded DNA probe linked to the cystic fibrosis locus to detect a single-copy gene from varying amounts of genomic DNA, with Southern blot analysis. The DNA plasmid probe was labeled with either biotin or digoxigenin. Biotin or digoxigenin was then linked to alkaline phosphatase (ALP) with use of streptavidin or anti-digoxigenin antibodies, respectively. ALP activity was then detected with a chromogenic (BCIP/NBT), chemiluminogenic (AMPPD), or fluorogenic (HNPP) substrate. Our results suggest that biotin and digoxigenin perform similarly and that the three substrates exhibit similar detectability under appropriate substrate incubation times: 20-120 min (AMPPD), 2-12 h (HNPP), and 18-48 h (BCIP/NBT). Under optimised conditions and the probe used, these methods detect single-copy genes from as little as 0.3 micrograms of total genomic DNA.

Biotin↗

PubMed1992

Quantitative western blot analysis and spot immunodetection using time-resolved fluorometry.

We describe a new method of staining and quantification of proteins blotted or spotted on nitrocellulose. Blotted or spotted proteins are first reacted with specific antibodies followed by reaction with biotinylated secondary antibodies. The immunocomplex is then reacted with a streptavidin-based macromolecular complex labeled with the fluorescent europium chelate of 4,7-bis(chlorosulfophenyl) 1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). The fluorescent spots or bands can then be assessed by visual inspection under UV illumination, by instant photography or quantified by scanning with a time-resolved fluorometer. The method does not involve enzyme detection, is simple, sensitive and gives sharp bands which remain fluorescent for long periods of time (months to years).

Animals↗