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loveskyzhou

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The supernatant was loaded onto a
glutathione sepharose column (GE Healthcare). After extensive
washing with 20mM Tris buffer pH 8.0, 500mM NaCl, 1mM DTT,
the GSTmoiety was cleaved by His-tagged TEV protease in the same
wash buffer at 4¡ãC overnight. The protein was passed through a Ni2+-
NTA agarose column (Qiagen) to eliminate the His-tagged TEV
protease,
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cloverplm

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silicare(½ð±Ò+5, BioEPI+1): 2011-08-10 12:05:50
loveskyzhou(½ð±Ò+2): лл 2011-08-10 12:55:27
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·Ö×°ÉÏÇåÖÁ¹Èë׸ÊëÄÊÕ¼¯Öù(GE Healthcare)£¬ÓÃ20mM Tris buffer pH 8.0, 500mM NaCl, 1mM DTT³ä·ÖÏ´µÓºó£¬ÔÙÓÃÏàͬµÄbuffer4¡æ½þÅݹýÒ¹£¬²¿·ÖGST±»His-tagged TEV µ°°×ø¼ôÇС£Ëæºó¸Ãµ°°×ͨ¹ýNi2+- NTA ÇíÖ¬ÌÇ·ÖÀëÖù£¨Qiagen£©È¥³ýHis-tagged TEV µ°°×ø¡£
2Â¥2011-08-09 20:58:15
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imfly77

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adslye(½ð±Ò+5): ½âÊ͵غÜÇå³þÁË£¬¸Ðл½»Á÷£¡ 2011-08-29 09:53:30
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2Â¥: Originally posted by cloverplm at 2011-08-09 20:58:15:
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·Ö×°ÉÏÇåÖÁ¹Èë׸ÊëÄÊÕ¼¯Öù(GE Healthcare)£¬ÓÃ20mM Tris buffer pH 8.0, 500mM NaCl, 1mM DTT³ä·ÖÏ´µÓºó£¬ÔÙÓÃÏàͬµÄbuffer4¡æ½þÅݹýÒ¹£¬²¿·ÖGST±»His-tagged TEV µ°°×ø¼ôÇС£Ëæºó¸Ãµ°°×ͨ¹ýNi2+- N ...

¼òµ¥µÄ˵£¬£¨Ä¿µÄµ°°×-GST£©Ê×ÏȽáºÏÓÚglutathione sepharose column£¬È»ºóͨ¹ý¼ÓÈëHis-tagged TEV protease £¨µ°°×ø£©ÇжÏÄ¿µÄµ°°×ºÍGSTÖ®¼äµÄÁ¬½ÓëÄ£¬ÔÙ¼ÓÈëbuffer¾Í¿ÉÒÔ°ÑÄ¿µÄµ°°×Ï´ÍÑÏÂÀ´£¬Öù×ÓÉÏÔòÊ£Óà½áºÏµÄGST¡£
Ï´ÍѳöµÄÄ¿µÄµ°°×ÀïÃæ»¹ÓÐHis-tagged TEV proteaseÔÓÖÊ£¬Õⲿ·Öͨ¹ýNiÖùºÍHis-tagged TEV proteaseÌØÒìÐÔ½áºÏ¶øÈ¥µô£¬×îºóÈÜÒºÖоÍÖ»ÓÐÄ¿µÄµ°°×ÁË¡£

Õâ¸ö·½·¨ÊÇĿǰÓõıȽ϶àµÄÒ»ÖÖ´¿»¯·½·¨£¬µ«Í¨¹ýÕâЩÇ׺ÍÎü¸½Ö®ÀàµÄ°ì·¨´¿»¯µ°°×ÖÊ£¬×îºó¶¼±ØÐ뽫ÖîÈçHistag¡¢GSTºÍCBDÖ®ÀàµÄ±ê¼ÇtagÈ¥µô¡£µ°°×ø·½·¨ÊÇʵÑéÊÒÀï³£ÓõÄÒ»ÖÖ¡£ÁíÍâÒ²ÓаüÀ¨»¯Ñ§·¨£¨ÀýÈçôǰ·ºÍä廯Ç⣩¡¢ÄÚº¬ëÄ£¨intein£©Ö®ÀàµÄµÈµÈÆäËûÊֶΡ£
3Â¥2011-08-10 15:27:51
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