Computerized analysis of double-label autoradiographs: A screening method for 1,25-dihydroxyvitamin D3 induced changes in intestinal protein synthesis
✍ Scribed by Nancy C. Kendrick; Kurt Eakle; Dr. Hector F. Deluca
- Publisher
- John Wiley and Sons
- Year
- 1982
- Tongue
- English
- Weight
- 936 KB
- Volume
- 3
- Category
- Article
- ISSN
- 0173-0835
No coin nor oath required. For personal study only.
✦ Synopsis
Abstract
The McIDAS computer system (Man‐computer Interactive Data Access System) at the University of Wisconsin was used to analyze double‐label autoradiographs of two‐dimensional (2‐D) electrophoresis gels. The 4 samples used to generate the 2‐D gels were obtained from 8 vitamin D‐deficient rat intestines 25 h after injection with 1,25‐dhydroxyvitamin D~3~ (1,25‐(OH)~2~ D~3~) or vehicle. To perform the analysis, the patterns on Kodak XAR5 (XR) film, which detects ^14^C + ^3^H by fluorography at ‐75 °C, and on Kodak NS‐2T (NS) film, which detects ^14^C only by direct autoradiography at room temperature, were converted to digital images. The McIDAS system was used to multiply the NS image by a normalization factor which matched it to the XR image with respect to exposure. After background subtraction from both images, the XR image was divided by the aligned normalized NS image; the resultant ratio image was multiplied by an enhancement factor and colored according to pixel brightness. Polypeptides with a high or low ratio relative to the average ratio stood out clearly in contrasting colors on the ratio image and could be detected by visual inspection. The actual XR/NS density ratios of these highlighted spots could then be quantitated by spot integration. To test the method, XR/NS density ratios were measured for 34 spots on each of the 4 films, sets and the spot ratios compared to the spot appearances on the ratio image. With the exception of a few false positives, agreement was good. Several polypeptides were found to be increased by 25–50 % relative to the average ratio while one polypeptide of molecular weight 50 000, approximate pI 5.5, was increased an average of 240 %.