2 replies to this topic

[tpd79]

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

[Nabin]

You can normalize baased on protein amount. Do protein assay and start with same amount of proteins for different cell lines.

[bob1]

Freeze-thaw alone is usually sufficient to disrupt the nuclear membrane.