查看完整版本: 生物系同学请进来帮我一下

生物系同学请进来帮我一下

我现在的program是自己从未接触过的
所以需要生物系同学帮我指点一下

我现在需要一些最常见而且易于测定其活性的蛋白酶
这个信息昨天nana已经提供给我了，我正在NEB的资料里面选择
但是还有个问题就是如何测定这些protease的活性
首先需要进行定性测定
之后再是定量
我现在不知道该看些什么文献或者textbook

所以需要生物系的同学给我提供些信息

可以在下面跟帖留言
也可以告诉我你的电话或者email，我找你主动联系一下
最好能当面问一下

应该是测这种酶break peptide bonds的反应速度吧。
你去wikipedia搜一下吧。

Anal Biochem. 1979 Aug;97(1):11-6.  Links
A new method for protease activity measurement.
Sugiura M, Ishikawa M, Sasaki M, Hirano K, Ito Y, Awazu S.

NCBI有全文的pdf

Protease assay

In certain fruits, such as pineapples and mangoes, the flesh contains protein-digesting enzymes (proteases). These may play a part in helping to soften the fruit tissues as the fruit ripens, making it even more attractive to animals that might disperse the seeds. So perhaps the activities of these proteases enzymes will increase during the ripening process.

Applications of plant proteases, such as ‘bromelain’ from the stems and fruits of pineapples, include uses in the pharmaceutical industry as a blood anti-coagulant, and in the prevention of proteinaceous hazes in chill-proof beers!

The assay

Protease enzymes are added to a milky colloidal suspension of egg albumen. As the protease enzymes digest the suspended particles of proteins, the mixture becomes more transparent. The absorbance changes in the reaction mixture are followed with a colorimeter. The protease content, for example in extracts of fruits, can be assayed by measuring the rate at which the solution of egg albumen and extract becomes clearer.


Preparation of the egg albumen colloidal suspension (enzyme substrate)

Separate the white of a single egg into a 250 cm3 beaker and add 150 cm3 tap water, stirring the mixture thoroughly. The mixture becomes quite cloudy as a result of the denaturation of some of the egg albumen by the water.


Place the beaker on a tripod and gauze and heat with a bunsen until the mixture boils, stirring it regularly.


Allow the mixture to cool, then decant it through two or three layers of muslin into another beaker. This creates a homogenous milky colloidal solution.
Carrying out the reaction

Select a test-tube that fits into the colorimeter. Add 2 cm3 of an appropriate buffer (e.g. pH7), followed by 2 cm3 of the albumen substrate solution and 1 cm3 of fruit extract.


Mix the contents of the colorimeter tube, and place it in the colorimeter (previously zeroed using a tube with 4 cm3 buffer and 1 cm3 of fruit extract). Read the absorbance and note the time.


Place the reaction mixture(s) in a water bath at 30°C to promote the activity of any protease enzymes present.


Take further absorbance readings at regular time intervals (say every 5 or 10 minutes), until no further change (decrease) in absorbance is detected.


Plot a graph of the change (decline) in absorbance against time. Measuring the time taken for a 50% reduction in absorbance value gives an indication of the protease activity of the original fruit extract.

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Ideas for investigations with this system (with fruits and vegetables)

Follow the changes in protease enzymes during ripening


Compare the effects of pH on proteases from different fruits


Compare the effects of temperature on proteases from different fruits


Investigate the presence of protease-inhibitors in the seeds of legumes


Monitor the release of amino acids from the digested proteins, using paper chromatography

不过sigma好像有做好的kit卖

Protease Fluorescent Detection Kit, PF0100

我没查过，不过我觉得ncb的catalog上是不是会有protocol啊?
或者你决定买一种，完了，直接在ncbi里面搜做这种酶的文章，methods里面肯定有的

ncbi？
umass有没有购买它的文章？我能不能从图书馆里面down到啊？

大家先不告诉他，直到把坑填了为止。

I have downloaded it here, give me an emailaddress, I will send it to you
Umass really sucks...there are tons of journals they didn't buy


[quote]原帖由 [i]那一片俗[/i] 于 2007-1-25 09:51 PM 发表
ncbi？
umass有没有购买它的文章？我能不能从图书馆里面down到啊？ [/quote]