Thin Layer Chromatography of Gut Extracts from Adults and Larvae of Donacia versicolorea and Macroplea mutica

TLC plates to assess the enzymatic activity of pools of 3 guts of Donacia versicolorea (Dver) and Macroplea mutica (Mmut) larvae and adults. 51 μg of protein were incubated with 0.4% (w/v) of either Polygalacturonic Acid, Carboxymethyl Cellulose 4M or Xyloglucan in a 20 mM citrate/phosphate buffer pH 5.0 to a total volume of 20 μl at 40 °C for about 16 h. Samples were applied to TLC plates in 4 2.5 μl steps, and plates were developed ascending with ethyl acetate: glacial acetic acid: formic acid: water (9:3:1:4) for about 120 min.

After drying, carbohydrates were stained by spraying plates in 0.2% (w/v) orcinol in methanol: sulfuric acid (9:1), followed by a short heating until spots appeared. For each plate, samples are in the following order: adults x the 3 substrates (same order as mentioned) + no substrate control, larvae x the 3 substrates + no substrate control, standards for galacturonic acid (monomer, dimer and trimer) and glucose oligomers (from mono- to hexamer), followed by each of the substrates with no added gut extract.