Separation and microanalysis of growth factors by Phast system gel electrophoresis and by DNA synthesis in cell culture.
A simple, micro-scale method was established for the characterization of growth factors at picogram levels using Phast system gel electrophoresis followed by monitoring the mitogenic activity by DNA synthesis in cell culture instead of staining methods. The separations and bioassays were carried out with a procedure involving Phast polyacrylamide gel electrophoresis or isoelectric focusing, gel slicing along the template, elution of growth factors through Transwell membranes and measurement of [3H]thymidine incorporation into DNA of normal rat kidney (NRK) fibroblasts. Transwell cell culture chamber inserts separated sliced gel pieces from culture cells and also permitted the direct elution of growth factors into the culture medium. The lower limit of sensitivity for human epidermal growth factor (hEGF) and transforming growth factor type alpha (TGF-alpha) were about 50 and 200 pg, respectively. At these concentrations, they were not detectable by the current most sensitive silver staining technique. Iodinated hEGF and TGF-alpha were also used to demonstrate the feasibility of determining the isoelectric point and molecular weight of peptides at picogram levels. This method is reliable, reproducible and can improve current methods for the characterization of growth factors.
Kuo
K W
KW
Department of Biochemistry, Kaohsiung Medical College, Taiwan.
Yeh
H W
HW
Chu
D Z
DZ
Yeh
Y C
YC
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000032:analysis / Q000302:isolation & purification; D005346:Fibroblast Growth Factors / Q000032:analysis / Q000302:isolation & purification; D006133:Growth Substances / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D008832:Microchemistry; D008934:Mitogens; D008970:Molecular Weight; D010982:Platelet-Derived Growth Factor / Q000032:analysis / Q000302:isolation & purification; D051381:Rats; D016211:Transforming Growth Factor alpha / Q000032:analysis / Q000302:isolation & purification
D006133:Growth Substances; D008934:Mitogens; D010982:Platelet-Derived Growth Factor; D016211:Transforming Growth Factor alpha; D005346:Fibroblast Growth Factors; D004815:Epidermal Growth Factor; D004247:DNA