I am the primary author of this protocol and I have two suggestions regarding this protocol based on my experience implementing it in a different lab:

1. Hydrolysis time in the autoclave may need to be optimized, likely because of differences in the speed of cooling and exhausting. The polymer that is most recalcitrant to sulfuric acid hydrolysis is polygalacturonic acid (PG), and the released galacturonic acid is degraded during hydrolysis conditions as well. My initial experience was that a 1 hour hydrolysis was insufficient when working with Arabidopsis cell wall material and an autoclave that exhausted relatively quickly. Incomplete hydrolysis of PG yields oligomers that can be detected by HPAEC-PAD with a sodium acetate gradient, which can be helpful in optimizing hydrolysis conditions. The ratio of the peak area between the monomer and the dimer is a particularly good metric of the extent of hydrolysis: Initial experiments with 60 minute hydrolysis gave a ratio of 6, while 120 minutes of hydrolysis gave a ratio of 18. In these conditions, the correction factor for GalUA (0.54) has a relatively large impact on the quantification, underscoring that product degradation and extent of hydrolysis must be optimized in order to obtain good results. Commercial pectin and PG of know GalA content are useful for such optimization experiments.

2. Analysis of uronic acids can be performed on a CarboPac PA20 column using the same conditions indicated in this protocol or by isocratic elution with 170 mM sodium acetate, 100 mM NaOH. I have found the latter to be more convenient as the baseline does not increase during the run.
4/19/2017 10:15:04 AM Reply