DETERMINATION OF GLYCOGEN IN SMALL TISSUE SAMPLES
DETERMINATION OF GLYCOGEN IN SMALL TISSUE SAMPLES
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DOI：
10.1152/jappl.1970.28.2.234
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发表时间：
1970-01-01
影响因子：
3.3
通讯作者：
TAYLOR, AW
中科院分区：
文献类型：
--
作者：
LO, S;RUSSELL, JC;TAYLOR, AW
材料和方法试剂。1)饱和硫酸钠的30%氢氧化钾溶液。氢氧化钾颗粒(300g；默克73201号，试剂级)在蒸馏水中溶解至1升，并用硫酸钠饱和(费舍尔编号：73201)。S-421，无水，有认证的ACS)。2)95%的乙醇。3)5%的苯酚。苯酚晶体(250g；Mallinckrodt no.9928，试剂级)在蒸馏水中溶解至5升。4)硫酸96-98%(Fisher No.A-300，试剂级)。5)标准糖原溶液。糖原粉(25 mg；Fisher No.G-47(试剂化学)在蒸馏水中溶解至5毫升，糖原浓度为5 mg/毫升。将该原液进行体积稀释，制备浓度较低的标准糖原溶液。I)用活检针采集人体肌肉样本，分离出一块重约35-50毫克的肌肉。肌肉样本立即被转移到称重盘中，所有可见的脂肪、结缔组织和血液都被用探头、钳子和纱布去除。然后在罗勒-史密斯精密扭转天平上称重样品，并用钳子将其转移到有盖的试管的底部。样品和试管立即在干冰和酒精中冷冻。仍有血迹的平底锅再次称重，从上次重量中减去血液的重量，得出试管中样本的重量。样品被冷藏，直到准备好进行化验。2)从冷藏中取出管子后，将管子冰冻保存。向样品中加入0.5毫升饱和了Na2SOd的30℃KOH，以确保组织完全浸入溶液中。3)将上述带螺帽的管子放入沸水浴中20-30分钟，直至得到均匀的溶液。
MATERIALS AND METHODSReagents. 1) Thirty percent potassium hydroxide solution saturated with sodium sulfate. Potassium hydroxide pellets (300 g; Merck no. 73201, reagent grade) were dissolved in distilled water to 1 liter and saturated with sodium sulfate (Fisher no. S-421, anhydrous, certified ACS). 2) Ninety-five percent ethanol. 3) Five percent phenol. Phenol crystals (250 g; Mallinckrodt no. 9928, reagent grade) were dissolved in distilled water to 5 liters. 4) Sulfuric acid 96-98%(Fisher no. A-300, reagent grade). 5) Standard glycogen solutions. Glycogen powder (25 mg; Fisher no. G-47, reagent chemical) was dissolved in distilled water to 5 ml. This gave a glycogen concentration of 5 mg/ml. Less concentrated standard glycogen solutions were prepared by volumetric dilution of this stock solution.Procedure. I) Human muscle samples were taken, with a biopsy needle, isolating a piece of muscle weighing approximately 35-50 mg. The muscle samples were immediately transferred to a weighing pan, and all visible fat, connective tissue, and blood were removed with a probe, forceps, and gauze. The samples were then weighed on a Roller-Smith precision torsion balance and transferred with forceps to the bottom of a capped test tube. Immediately the sample and test tube were frozen in Dry Ice and alcohol. The pan, with traces of blood still remaining, was again weighed, and the weight of blood subtracted from the previous weight to give the weight of the sample in the test tube. Samples were maintained deep-frozen until ready for assay. 2) The tubes were kept on ice after removal from frozen storage. One-half milliliter of 30? & KOH saturated with Na2SOd was added to the samples, making sure that the tissue was completely immersed in the solution. 3) The above tubes with the screw cap on were put in a boiling water bath for 20-30 min until a homogeneous solution was obtained.