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 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.
Koyano
Y
Y
Department of Biochemistry, Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL 60612, USA.
Hämmerle
H
H
Mollenhauer
J
J
D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002645:Chickens; D003094:Collagen / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005374:Filtration; D006909:Hydroxyproline / Q000032:analysis; D011392:Proline / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate; D014238:Trichloroacetic Acid / Q000494:pharmacology; D014316:Tritium / Q000378:metabolism
D014316:Tritium; D012967:Sodium Dodecyl Sulfate; D014238:Trichloroacetic Acid; D003094:Collagen; D011392:Proline; D006909:Hydroxyproline