UDBS063 at UK.AC.KCL.CC.HAZEL wrote:
:: I am trying to isolate functioning chloroplasts from brown algae Laminaria.
: I succeeded in isolating chloroplasts, which looked O.K. but did not
: evolve oxygen. Can anybody advice me?
: I am using Trcine buffer (pH 7.5) with 0.3 M Sorbitol, 5mM EGTA,5mM EDTA,
: 1% BSA 5mM DTT, 5mM Ascorbate and 2% PVP as isolation medium.
Did you keep everything _cold_ during the isolation?
The best method is to work in a 'cold' room (4C max), pre-chill
everything and work quickly. Also, use a macerator that shears rather
than cuts (ie. don't disrupt the choloplasts mechanically).
.... and make sure your oxygen electrode is working :-)
Dr. A.J.Travis, | JANET: <ajt at uk.ac.sari.rri>
Rowett Research Institute, | other: <ajt at rri.sari.ac.uk>
Greenburn Road, Bucksburn, | phone: +44 (0)224 712751
Aberdeen, AB2 9SB. UK. | fax: +44 (0)224 715349