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小木虫论坛-学术科研互动平台 » 生物医药区 » 分子生物 » 综合其他 » 关于在表达蛋白和GFP蛋白之间的linker
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丫头7976

铜虫 (正式写手)

[求助] 关于在表达蛋白和GFP蛋白之间的linker

各位好，我最近在做膜蛋白的表达，在构建载体的时候我在膜蛋白的C端还加了GFP指示蛋白，但是在诱导表达后，根本看不到菌发出绿色荧光，而我只表达GFP时却能看到菌是绿色的，我在构建载体的时把膜蛋白和GFP直接连在一起了，中间没有加linker regions，我想问一下各位，我的菌株看不出绿色的原因是不是我没有加一个linker的原因呢，如果有做过这方面的大侠是不是可以知道一下我呢，多谢各位！
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1楼 2011-06-03 19:13:02
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bio-research

金虫 (职业作家)

【答案】应助回帖

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丫头7976(金币+3): Thank you for your advices，but 2011-06-06 17:42:47
dhd997(金币+5): good 2011-06-06 21:28:13
protein of interest surely weakens fluorescence of GFP with linker or without linker.
i have ever made experiment about expression of fusion protein including GFP,
the fusion protein including GFP can emit green fluorescence although its green fluorescence is weaker than that of control.
but the fusion protein in my experiment is located at the cytoplasm, and your protein occured in the membrane, I don`t know whether location of protein including GFP effects fluorescence emission or not.
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2楼2011-06-06 11:13:02
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丫头7976

铜虫 (正式写手)
引用回帖:
Originally posted by bio-research at 2011-06-06 11:13:02:
protein of interest surely weakens fluorescence of GFP with linker or without linker.
i have ever made experiment about expression of fusion protein including GFP,
the fusion protein including GF ...

Thank you very much for your advice, but there is a question, when you express fusion protein including GFP,  can you watch the green fluorescence with the naked eye ?
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3楼2011-06-06 17:47:22
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bio-research

金虫 (职业作家)

【答案】应助回帖

丫头7976(金币+1): thanks 2011-06-07 16:11:20
引用回帖:
Originally posted by 丫头7976 at 2011-06-06 17:47:22:
Thank you very much for your advice, but there is a question, when you express fusion protein including GFP,  can you watch the green fluorescence with the naked eye ?

no,I can`t.
even green fluorescence wasn`t seen in the control group(only GFP) by naked eyes.
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家有小子初长成!(Nothing is impossible to a willing heart!)
4楼2011-06-06 17:59:40
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西瓜

荣誉版主 (知名作家)

【答案】应助回帖

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丫头7976(金币+2): thanks 2011-06-07 16:09:05
dhd997(金币+2): 还拽上了 2011-06-07 16:51:12
引用回帖:
Originally posted by 丫头7976 at 2011-06-06 17:47:22:
Thank you very much for your advice, but there is a question, when you express fusion protein including GFP,  can you watch the green fluorescence with the naked eye ?

You can't see the fluorescence if the GFP is not excitated. But, if your fusion protein is well expressed, you can see that the bacteria expressing the fusion protein is green, without excitation of GFP. If you purify the fusion protein, the solution containing the protein is also green, very lovely

I've seen those with my own eyes. So, trust me

[ Last edited by 西瓜 on 2011-6-6 at 22:48 ]
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5楼2011-06-06 22:35:00
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丫头7976

铜虫 (正式写手)
引用回帖:
Originally posted by 西瓜 at 2011-06-06 22:35:00:
You can't see the fluorescence if the GFP is not excitated. But, if your fusion protein is well expressed, you can see that the bacteria expressing the fusion protein is green, without excita ...

thank you for your help, I'd like to make a quest if I may,could you give me  a QQ number? I want to make a more specific communications. I hope you will meet.
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6楼2011-06-07 16:18:04
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西瓜

荣誉版主 (知名作家)
引用回帖:
Originally posted by 丫头7976 at 2011-06-07 16:18:04:
thank you for your help, I'd like to make a quest if I may,could you give me  a QQ number? I want to make a more specific communications. I hope you will meet.

不客气啊
你是要偶QQ还是要我帮你申请个QQ？
反正都ok……站内短消息联系我就好。
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7楼2011-06-08 23:11:27
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丫头7976

铜虫 (正式写手)
★
dhd997(金币+1): 欢迎来本版继续交流 2011-07-17 09:42:46
引用回帖:
Originally posted by 丫头7976 at 2011-06-07 16:18:04:
thank you for your help, I'd like to make a quest if I may,could you give me  a QQ number? I want to make a more specific communications. I hope you will meet.

你好，我最近又在做膜蛋白与GFP的融合蛋白，我诱导表达后，用肉眼看不出菌体是绿色的，是我的蛋白的表达量少看不出绿色呢，还是说得用激发光激发才能看出绿色呢，你有做过膜蛋白与GFP融合蛋白的表达吗，确定能用肉眼看出菌体是绿色的吗，请用中文回复我吧，英文交流的不是很清楚，谢谢你啦
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8楼2011-07-16 21:13:56
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mybrony

银虫 (正式写手)
这个问题我也遇到了，linker帮助不大哦，我试过的
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9楼2011-11-29 18:39:53
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roomofxw

木虫 (正式写手)
我随便敲敲键盘的啊
SDS-PAGE确定GFP是否表达了，或者融合蛋白表达量很低
膜蛋白具有疏水区域，倾向于形成聚集体，聚集有可能造成GFP功能丧失，甚至性成包涵体。
双阅读框共表达目的蛋白和GFP，确认目的蛋白特性是否对GFP有影响。
另外我做过大肠和酵母表达GFP，肉眼是可以观察到绿色菌体
但是如果表达量比较低的情况下，我认为还是需要荧光显微镜的
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10楼2011-11-30 11:47:31
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