试验原理
如图所示,糖苷酶水解底物无色ONPG,生成黄色产物,通过测量吸光值,可以得出糖苷酶的活性。
所用试剂
Z buffer, per 50 mL:
* O.80g Na2HPO4.7H2O (0.06M)
* 0.28g NaH2PO4.H2O (0.04M)
* 0.5 mL 1M KCl (0.01M)
* 0.05 mL 1M MgSO4 (0.001M)
* 0.135 mL b -mercaptoethanol (BME) (0.05M)
* bring to approximately 40 mL with H2O, dissolve all the salts
* adjust the pH to 7.0
* use a graduated cylinder to bring the buffer to 50 mL
* donot autoclave, store at 4 C.
ONPG
ONPG should be dissolved fresh each day. 可以溶于PBS,Z buffer或者水中。
PBS
Stop solution
1M Na2CO3
操作步骤
准备样品
将菌培养至稳定期(OD600约为2)
按照1:50的比例转接到新鲜培养基中(并按时间梯度依次取样)
样品离心后,用PBS冲洗,然后保存在-80摄氏度待用
测量酶活
将细胞用Z buffer重悬
然后超声破碎裂解细胞或者加入氯仿和SDS裂解
将生色底物 2-硝基苯-beta-D-半乳糖苷加入反应液中
在28摄氏度下孵育1小时
加入终止液(1M Na2CO3)终止反应
取溶液上清,测量420nM和550nM下的吸光度
计算过程
则糖苷酶活性(Miller单位)=(1000 X A420) / (time [min] X 样品体积
[ml] X A600)。
另外一种计算公式(使用550nM吸光值作为修正)
Miller Units =
1000 x [(OD420 – 1.75 x OD550)] / (T x V x
OD600)
- OD420 and OD550 are
read from the reaction mixture. - OD600 reflects cell density in
the washed cell suspension. - T = time of the reaction in
minutes. - V = volume of culture used in the assay in
mLs.
The units give the change in A420/min/mL of
cells/OD600
![](https://cloudfs-spring.oss-cn-qingdao.aliyuncs.com/bio_spring_uploads/2011/02/2011-02-16-203553_648x613_scrot.png)
Beta-Galactosidase Activity Assay. at <http://rothlab.ucdavis.edu/protocols/beta-galactosidase-3.html