Analysis of 3H-proline-labeled protein by rapid filtration in multiwell plates for the study of collagen metabolism
Quantitative and qualitative analysis of cell cultures labeled with 3H-proline for monitoring collagen synthesis is time-consuming and occasionally generates large quantities of radioactive waste. This present work describes the application of a microwell filtration system for the analysis of collag...
| Veröffentlicht in: | BioTechniques. - 1991. - 22(1997), 4 vom: 21. Apr., Seite 706-8, 710-2, 714 passim |
|---|---|
| 1. Verfasser: | |
| Weitere Verfasser: | , |
| Format: | Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
1997
|
| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Journal Article Research Support, U.S. Gov't, P.H.S. Tritium 10028-17-8 Sodium Dodecyl Sulfate 368GB5141J Trichloroacetic Acid 5V2JDO056X Collagen 9007-34-5 mehr... |
| Zusammenfassung: | Quantitative and qualitative analysis of cell cultures labeled with 3H-proline for monitoring collagen synthesis is time-consuming and occasionally generates large quantities of radioactive waste. This present work describes the application of a microwell filtration system for the analysis of collagen metabolism in chondrocytes. It is based on trichloroacetic acid (TCA) precipitation of 3H-proline-labeled proteins onto polyvinylidene difluoride membranes fitted in a 96-well plate and subsequent analysis of precipitated proteins by liquid scintillation counting, amino acid analysis and sodium dodecyl sulfate (SDS) gel electrophoresis. This method allows for the initial processing of 96 samples within 2 h, has high sensitivity and accuracy (linearity > or = 200 cpm/sample) for quantitative measurements and a capacity of up to 10 micrograms collagen/microwell. The ratio of the radioactive protein collected by this filtration assay compared to that collected by molecular sieve chromatography on Sephadex G-25 was linear over a broad range, indicating full compatibility of data and a high reproducibility for both assay systems. The quality of protein separation by SDS-polyacrylamide gel electrophoresis (PAGE) from samples obtained by the filtration assay and by dialysis was virtually identical. These features make the assay particularly suited for pulse-chase experiments and for monitoring protein degradation |
|---|---|
| Beschreibung: | Date Completed 08.07.1997 Date Revised 21.11.2013 published: Print Citation Status MEDLINE |
| ISSN: | 1940-9818 |