Radioimmunoassay of Gibberellins A5 and A20

Distribution of Gibberellins and Expressional Analysis of GA 20-oxidase Genes of Morning Glory during Fruit Maturation

“…14) GA 20 was similarly analyzed using anti-GA 20 -Me-antiserum. 15) An immunoreactive peak attributable to GA 1=3 from its t R (23-24 min) on HPLC was clearly observed in extracts both from sample A and sample B of 6-12 DAA (Fig. 2B) when analyzed with the anti-GA 1 -Meantiserum, while an apparent immunoreactive peak attributable to GA 20 (t R 25-26 min) was observed only in the extract from sample B when analyzed with the anti-GA 20 -Me-antiserum.…”

“…7) Each fraction was methylated with diazomethane, dried in vacuo, dissolved in 1 ml of 5% (v/v) methanol, and subjected to radioimmunoassay (RIA). The procedure for RIA was as follows: (i) 100 ml of fractionated solution described above was added to the glass tube for RIA; (ii) 400 ml of 1/10-diluted bovine serum solution in phosphate-buffered saline (PBS) containing tritium-labeled GA 4 -Me or tritium-labeled GA 20 -Me (166 bq per assay, tritium-labeled GA 4 -Me was used for the quantification of active GA 1=3 and tritium-labeled GA 20 -Me for that of GA 20 ) was added to the tube; (iii) 100 ml of 1/20,000-diluted anti-GA 1 -Me antiserum 14) in PBS or 1/5,000-diluted anti-GA 20 -Me antiserum 15) in PBS was added to the tube; (iv) incubation at 4 C overnight after vigorous mixing; (v) 750 ml of saturated ammonium sulfate was added to the tube and mixing followed by incubation at 4 C for 30 min; (vi) centrifugation (2;000g, 20 min, 4 C) and discarding of the supernatant; (vii) 1 ml of 50% (v/v)-saturated ammonium sulfate was added to the tube and mixing; (viii) centrifugation (2;000g, 20 min, 4 C) and discarding of the supernatant; (ix) 100 ml of water and 1 ml of scintillator (ACSII scintillation cocktail, Amersham Biosciences, NJ) were added to the tube and evaluation of radioactivity with a scintillation counter (Aloka, Tokyo, Japan). C linear gradient at 15 C min À1 ; He flow: 1 ml min À1 ; ionization: EI (70 eV).…”

“…The metabolite(s) of GA 12 were methylated with CH 2 N 2 and subjected to GC/MS, and those of GA 53 were methylated and trimethylsilylated prior to GC/MS. GA 15 and GA 44 were identified by comparing their MS and Kovats retention indices 16) to authentic ones.…”

“…On the other hand, we have noticed that the content of GA 20 in the growing fruit of morning glory rapidly increased at stages later than 16 DAA. 15) The rapid increase in GA 20 in the fruit at later stages suggests that biosynthesis of GA 20 is induced in tissues other than the seed coat when cotyledons are well formed. The site where the signal for InGA20ox1 gene expression began to appear is thought to be the influx port of assimilation products.…”

Gibberellins and Antheridiogens in Prothallia and Sporophytes ofAnemia phyllitidis

“…Immunization was performed by the method of Yamaguchi l't al. 13 ) The immunogen (lV-antheridic acid-BSA. I mg) was dissolved in 0.6 ml of phosphate butfer saline (PBS; (U)I~I phosphate.…”