Things are moving along here with my cell wall samples. I performed acid hydrolysis to investigate the monosaccharide content of each sample (using only a small portion of the sample and keeping the rest intact for the protocol described below). These samples represent several different genomes of maize as well as different parts of the plant. Acid hydrolysis is a common method for breaking down complex carbohydrates into the monomers that they are composed of. I then used high performance liquid chromatography (HPLC) to analyze these monomers; this is a method that separates, with higher precision than a conventional column, compounds based on their polarity. These preliminary results gave us an idea of which samples could potentially be interesting—that is, which samples were unusual or were outliers compared to the others.
I am now moving forward with the potentially interesting samples to do the next part of the cell wall fractionation protocol. This will separate and isolate the different polymer components of the cell walls, such as the pectin, cellulose, and hemicellulose. We can then analyze, using chromatography methods, these polysaccharides individually and investigate their composition and structure more precisely. This part of the protocol is similar in concept to what I did to isolate the cell wall: it consists of many, many washes with a series of progressively “harsher” solvents. The compounds that are easier to extract—that is, those bound less strongly to the cell wall structure—come off into the supernatant sooner, while those that are bound more tightly only dissociate with the harshest solvents. So far I’ve extracted some of the pectins using ammonium oxalate, and more of the pectins as well as some lignin with sodium chlorite and acetic acid. Next, I’ll do several washes with sodium hydroxide, with the molarity increasing each round. Between each solvent I have to wash the samples exhaustively with water and dry them overnight, so it’s a time-consuming process. But it’s well worth it, because after it’s done I’ll be able to get some exciting results about the cell wall architecture of these samples!