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.

本实验采用薄层色谱（Thin Layer Chromatography, TLC）板，用于检测异色萤叶甲（Donacia versicolorea, Dver）与菱角萤叶甲（Macroplea mutica, Mmut）幼虫及成虫的3个肠道混合样品的酶活性。取51 μg蛋白质与0.4%（质量体积比，w/v）的多聚半乳糖醛酸（Polygalacturonic Acid）、羧甲基纤维素4M（Carboxymethyl Cellulose 4M）或木葡聚糖（Xyloglucan）中的一种，置于20 mM柠檬酸盐-磷酸盐缓冲液（citrate/phosphate buffer，pH 5.0）中，于40 ℃条件下孵育反应约16 h，反应总体积为20 μL。将样品分四次，每次2.5 μL点样于TLC板，随后采用上行展开法，以乙酸乙酯:冰乙酸:甲酸:水(9:3:1:4)为展开剂，展开约120 min。晾干薄板后，采用0.2%（w/v）地衣酚（orcinol）的甲醇-浓硫酸（9:1）溶液喷雾染色，随后短暂加热至糖类斑点显现。每块薄板的样品点样顺序如下：成虫对应3种底物（按前述顺序）+ 无底物对照，幼虫对应3种底物+无底物对照，半乳糖醛酸（galacturonic acid）标准品（单体、二聚体、三聚体）与葡萄糖寡聚体（glucose oligomers）标准品（从单糖到六糖），随后为各未添加肠道提取物的底物空白对照。