*4.8. Cell Wall Sugar Content Analysis*

The cellulose content of CWs was quantified by the Updegraff method [103], under the hydrolytic conditions described by Saeman, [104], using glucose as standard.

The total sugars and uronic acids were determined by the phenol-sulfuric acid method [105], and the m-hydroxydiphenyl method [106], using D-glucose and galacturonic acid as reference, respectively. For the neutral sugar estimation, the values for total sugars and uronic acids were subtracted.

For the neutral sugar composition, samples from each fraction were hydrolyzed with 2 N TFA for 1 h at 121 ◦C, which resulted in monosaccharides that were derivatized to alditol acetates following the method described by Albersheim [107]. Furthermore, the alditol acetates were quantified by gas chromatography (GC) using a Perkin-Elmer equipment with a flame ionization detector (GC-FID), using a Supelco SP-2330 column and a Perkin-Elmer GC-FID, as described in Rebaque et al. (2017) [61]. Inositol was used as internal control, and monosaccharides L(−)rhamnose (Merck), L(−)fucose (Sigma), L(+)arabinose (Merck), D(+)xylose (Merck), D(+)mannose (Merck), D(+)galactose (Merck), and D(+)glucose (Panreac) as standard markers.

#### *4.9. Gel Permeation Chromatography*

The CW polysaccharides were size-fractionated by using a sepharose CL-4B column in a 120 mL bed-volume (1.5 cm diameter) column in pyridine/acetic acid/water (1/1/23 *v*/*v*/*v*) at 0.3 mL/min. The column was calibrated with sucrose and different commercial dextrans of known relative average molecular weight (Mw) as described by Kerr and Fry [65]. By using the *K*av(1/2) method [65], a calibration curve was obtained (log Mw = −5.333 *K*av(1/2) + 8.103). V0 and Vi (*K*av 0 and 1) were defined by dextran blue (2000 kDa) and sucrose, respectively. Then, the polysaccharides were loaded into the column at a concentration of 100 μg/mL, and the total sugars were estimated for each fraction. The nominal Mw for each fraction was determined (nominal rather than absolute due to the conformational differences between dextran standards and CW polysaccharides).

#### *4.10. Immunodot Assays*

IDAs were carried out as described by García-Angulo et al. (2006) [89]. The reference compounds were commercial pectin (P41) and arabic gum. Monoclonal antibodies (mAbs) LM1, LM2, JIM5, JIM7, LM5, LM6, LM11, and LM15 were used with mung bean as standard. The reference compounds, as well as samples of fractions, were diluted 1/5, five times. Then aliquots of 1 μL from each dilution were spotted on nitrocellulose membranes. After drying, the membranes were blocked with 0.14 M NaCl, 2.7 mM KCl, 7.8◦ mM Na2HPO4.12H2O and 1.5◦ mM KH2PO4, pH 7.2 (PBS), with 4% fat-free milk powder, during 1.5 h at room temperature and incubated in primary antibody (hybridoma supernatants diluted 1/10). After washing the membranes, they were incubated in secondary antibody (antirat horseradish peroxidase conjugate, Sigma) diluted 1/1000, during 1.5 h at room temperature. The color was developed with 25 mL deionized water, 5 mL methanol with 10 mg mL−<sup>1</sup> 4–chloro–1–naphtol, 30 μL 6% (*v*/*v*) H2O2, and stopped by washing the membranes. A value ranging from 0 to 5 was assigned depending on the number of colored spots that were shown after developing the membranes as described in De Castro et al. (2014) [108]. As it corresponded with dilutions, this was used to establish a semiquantitative scale which was processed in a Heatmap, and clustering was performed with R [101].
