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植物组织样本的前处理
点击次数:202 发布时间:2015-8-12
一、匀浆介质
一般采用0.05 mol/L Tris-HCl, pH 7.4 磷酸盐缓冲液(PBS),客户可根据样本及测定指标的情况自行设定浓度,目的是保持样本的等渗环境。
二、组织匀浆的制备
1、取组织块(0.2g~0.5g)zui少可到5~10mg在冰冷的PBS中漂洗,滤纸拭干,准确称重,放入5ml的匀浆管中。
2、按重量(g):体积(ml)=1:4的比例加入4倍体积的匀浆介质于匀浆管中,冰水浴条件下,用眼科小剪尽快剪碎组织块。
3、匀浆的方式:手工匀浆,机器匀浆。
① 手工匀浆:左手持匀浆管将下端插入盛有冰水混合物的器皿中,右手将捣杆垂直插入套管中,上下转动研磨数十次(6~8分钟),充分研碎,制成20%的匀浆液。
② 机器匀浆:用组织捣碎机10000~15000 转/分 上下研磨制成20%组织匀浆,也可用内切式组织匀浆机制备(匀浆时间10秒/次,间隙30秒,连续3~5次,在冰水中进行,可适当延长匀浆时间),
镜检观察:取少量组织匀浆作涂片(直接涂片、染色均可以),显微镜下观察细胞是否破碎,若没有则可延长匀浆时间。
4、将制备好的20%匀浆液用普通离心机或低温低速离心机4000转/分左右,离心10~15分钟,取上清液进行测定.
三、样本保存:
植物组织样本暂时不测定,可立即低温冻存,温度越低越好,中间如*冻融,-20℃以下可保存三个月,-70℃以下可保存六个月。
制备好的匀浆液建议不要冻存,当天进行测定,如放置时间过长相关酶活会有所下降,部分指标4℃可存放3~5天(如SOD要存放2~3天,MDA可存放3~5,总蛋白测定可存5~7天等)
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