I'd inserted a glucanase gene into rice an would like to quantify its effect on cellulose, hemicellulose, matrix polysaccharide composition in rice leaf.
1) Which is the best method to quantify cellulose, hemicellulose, matrix polysaccharide composition in rice leaf? HPLC GCMS or HPAEC-PAD?
2) When preparing AIR sample (alcohol insoluble residue), i freeze dry the sample, wash with 70% ethanol, followed by washing with chloroform: methanol. Then add acetone vortex, centrifuge, discard acetone and air dry on bench overnight. Do i need to go for starch removal using amylase and pullulanase?
Any opinions and suggestions are welcomed and appreciated.
Thank you.
Dear Wong Ling Chie
Many thanks for your question. I cannot comment on the GC-part of your question so that I will leave it to the real experts in GC. Regarding the HPAE-PAD part l would like to share an article (see below) that is nicely summarizing the goes and no-goes with HPAE.
HPAE will allow you to separate polysaccharides with a DP > 100, with a maximum size around ~ DP 150. Any polysaccharide larger than that will be retained by the anion exchanger, not eluting.
If you are interested in analyzing larger polysaccharides than this, Size Exclusion Chromatography is a great tool. While SEC often is combined with RI-detection you might want to look into the second document I attached. It shows the combination of SEC and pulsed amperometric detection, hyphenating the best of both worlds.
My guess: if you are after the larger polysaccharides, and don't want to do a hydrolysis step, then I would look into SEC-PAD for its capability to give you some MW-distribution information with the benefit of a highly selective detection.
I hope this is of interest.
Good luck with your research,
Detlef.
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