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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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丫头7976

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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

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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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bio-research

金虫 (职业作家)

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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

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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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