I'm looking at GAG quantification from cell cultures using either DMMB or Alcian blue however I really need to normalise across different cell lines so would like to normalise to DNA content. Would a proteinase K digest (in 100mM ammonium acetate) followed by freeze thaw (1 or more cycles) be sufficient to release the cellular DNA so I can split the sample for quantification by picogreen without further processing and for GAG analysis? I don't really want to add other lysis agents as they may interfere with the GAG analysis.
Suggestions welcomed.
Thanks
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2 replies to this topic
#2
Nabin
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Posted 24 August 2012 - 01:05 PM
You can normalize baased on protein amount. Do protein assay and start with same amount of proteins for different cell lines.
#3
bob1
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Thelymitra pulchella
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Excellent
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Posted 24 August 2012 - 01:11 PM
Freeze-thaw alone is usually sufficient to disrupt the nuclear membrane.
Also tagged with one or more of these keywords: lysis, proteinase k, DNA
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