Re: [問題] 請益 zymography 的相關原理或方法
※ 引述《sheepfeather (...)》之銘言：
: 讀paper看到的新名詞 想搞懂他是啥技術
: 網路上資料不是很清楚 kiwi也說得很模糊
: 希望有人願意跟我說明一下基本原理或操作流程之類的
: 或是要去哪裡才可以查詢到相關資料都可以
: 感謝願意回答的各位 ~ <(_""_)>
Zymography is an electrophoretic technique, based on SDS-PAGE, that
includes a substrate copolymerized with the polyacrylamide gel, for
the detection of enzyme activity.
Samples are prepared in the standard SDS-PAGE treatment buffer but
without boiling, and without a reducing agent.
Following electrophoresis, the SDS is removed from the gel (or zymogram)
by incubation in unbuffered Triton X-100, followed by incubation in an
appropriate digestion buffer, for an optimized length of time at 37°C.
The zymogram is subsequently stained (commonly with Amido Black or Coomassie
Brilliant Blue), and areas of digestion appear as clear bands against a
darkly stained background where the substrate has been degraded by the enzyme.
These protocols, however, are subject to much adjustment. For instance,
D. melanogaster digestive glycosidases generally survive reducing conditions
(i.e. the presence of 2-mercaptoethanol or DTT) and and to an extent heating.
Indeed, the separations following heating to 50°C tend to exhibit a
substantial increase in band resolution, without appreciable loss of activity.
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