Quantitative western blotting using [gamma-33P]ATP and the ultrasensitive bio-imaging analyzer

This report describes a method for simultaneous quantitative determination of antigens from protein blots based on the highly sensitive "contact-copy" procedure, applying the phosphorylation reaction as a general detection principle. Using ultrasensitive bio-imaging analyzer systems permits us to quickly and easily quantify single bands directly from the picture screen and to achieve a high-resolution printing of the image (pictography). By applying a new kanamycin loading procedure it is possible to use phosphocellulose paper P81 as a substrate matrix. Replacing 32P with 33P as a detection isotope leads to an improvement of the sensitivity, resolution, and safety. The method is applied to analyze the proteins dystrophin, myosin, vinculin, and desmin from human tissue lysates. The high sensitivity of the procedure (detection limit approximately 1 pg dystrophin) permits determination of the quantity of dystrophin in very small tissue biopsy samples, which is of special interest for Duchenne/Becker muscular dystrophy diagnosis.