apoptosis gel electrophoresis
ZBIGNIEW DARZYNKIEWICZ (darzynk@nymc.edu)
Tue, 19 Sep 1995 09:41:32 -0500
Dr. Ross Longley poses a qestion why when using pulsed field gel
electrophoresis he cannot see multimers of the nucleosomal DNA
fragments.
Mononucleosomal DNA and multimers (up to 20 x 200 bp) are generally
detected by traditional agarose gel electrophoresis and not by pulsed
field electrophoresis: they migrate too fast and cannot be seen
(escape from gels) during pulsed field electrophoresis. Pulsed field
electrophoresis is used to detect very large fragments of DNA (50 -
300 kilobase; size of DNA loops in chromatin). Such fragments are
rarely detected during normal apoptosis which proceeds with
internucleosomal DNA cleavage. In some cell systems, however,
apoptotic DNA degradation stops at 300 - 50 kb and then pulsed field
electrophoresis can be used. Experimentally, DNA degradation can be
stopped at 50-300 kb DNA size by serine protease inhibitor TPCK (10
micromolar, added at the same time when inducer of apoptosis is
added). We are using the model system of HL-60 cells triggered to
apoptosis by 0.15 uM camptothecin; when camptothecin is added
together with 10 uM TPCK DNA degradation stops at 50 kb. This is a
convenient positive control cell system to study large moilecular
weight DNA degradation during apoptosis.
Zbigniew Darzynkiewicz
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