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Chloroplast isolation

Tony Travis ajt at rri.sari.ac.uk
Fri Mar 5 21:44:00 EST 1993

UDBS063 at UK.AC.KCL.CC.HAZEL wrote:
: Hi, 
: 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.
: Thanks

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

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