Glucose pap liquiform kit

This analysis considered a glycogen purification and enzyme digestion method to obtain glucose [24 (link)]. Briefly, 400 μl of 30% KOH were added to 100 mg of tissue, followed by incubation at 100°C for 15 min with agitation. A 50 μl sample of each homogenate were placed on a 1 × 1 cm Whatman 31 ET filter paper. A calibration curve with glycogen (2.4 mg/ml) was prepared. Glycogen was precipitated with 66% cold ethanol for 10 min while stirring. Then, two washes were performed with 66% cold ethanol and the papers were dried to remove ethanol. Afterwards, 1 ml of amyloglucosidase (Sigma, 10113) solution 0.5 mg/ml in 400 mM sodium acetate buffer pH 4.8 was added and was incubated for 2 h at 37°C in a stirred thermoregulated bath. Afterwards, 50 μl were extracted from each tube for the determination of glucose using the Glucose PAP liquiform kit (Labtest, Diagnóstica S.A.) and spectrophotometric assay by glucose oxidase and Trinder reaction, in which the product is a quinoneimine dye, which absorbs maximally at 505 nm [25 (link)]. Finally, it was taken to initial glycogen concentration through stoichiometry.

Sodium bisulfate, copper (ii) sulfate, potassium sodium tartrate tetrahydrate, tannic acid, Folin-Ciocalteu, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, sodium acetate, HEPES, minimum 99.5% titration, 2,4,6-tris (2-pyridyl)-5-triazine (TPTZ), methyl viologen dichloride hydrate (Paraquat), amyloglucosidase from Aspergillus niger, and 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl free radical (DPPH) were purchased from Sigma-Aldrich (São Paulo, SP, Brazil). Glucose PAP liquiform (Kit) was purchased from Labtest (Belo Horizonte, MG, BRA) and potassium ferrocyanide trihydrate, zinc acetate dihydrate, iron(II) ammonium sulfate hexahydrate, gallic acid monohydrate, aluminum chloride hexahydrate, potassium persulfate, and iron(III) chloride were purchased from Vetec Química Fina LTDA (Rio de Janeiro, RJ, BRA). All the other chemicals used in this work were of analytical grade.