GST-tagged protein purification by Glutathione Sepharose 4B遇到的难题# Biology - 生物学
a*z
1 楼
求建议。
一篇paper，老板什么也没干，只是加名字当2作，需要要求他当通讯作者吗？
听人说通讯作者搞个senior一点的审稿不会欺负人。
一篇paper，老板什么也没干，只是加名字当2作，需要要求他当通讯作者吗？
听人说通讯作者搞个senior一点的审稿不会欺负人。
a*e
2 楼
http://list.video.baidu.com/yingbang.html
用了这我就能在美国这看中国的电视吗？
用了这我就能在美国这看中国的电视吗？
f*s
3 楼
最近用GE healthcare的glutathione sepharose 4B resin purify a couple of GST-
tagged transcription factor DNA binding domains. The protein were bound to
the beads with high efficiency. However, when I used glutathione buffer to
elute the GST-protein off the beads, they can't be eluted. I tried 10 mM ,50
mM and 100 mM glutathione. To make the buffer, I diluted 1 M Tris (pH 7.5
at room temperature) to 50 mM, and added glutathione powder. There was also
2 mM DTT in the buffer. The elution was carried out at 4 degree where Tris
should be at pH 8.0.
I tried different resin and different glutathione, but none of them worked.
So the problem must be in the buffer conditions.
Does anyone know what is the potential problem? pH? I never measured pH of
the glutathione-Tris buffer.
tagged transcription factor DNA binding domains. The protein were bound to
the beads with high efficiency. However, when I used glutathione buffer to
elute the GST-protein off the beads, they can't be eluted. I tried 10 mM ,50
mM and 100 mM glutathione. To make the buffer, I diluted 1 M Tris (pH 7.5
at room temperature) to 50 mM, and added glutathione powder. There was also
2 mM DTT in the buffer. The elution was carried out at 4 degree where Tris
should be at pH 8.0.
I tried different resin and different glutathione, but none of them worked.
So the problem must be in the buffer conditions.
Does anyone know what is the potential problem? pH? I never measured pH of
the glutathione-Tris buffer.
t*b
4 楼
出来的时候详细看了一下介绍，和chromecast是一类的东西。
除非比chromecast还便宜很多，否则应该什么太大市场。
除非比chromecast还便宜很多，否则应该什么太大市场。
e*s
5 楼
如果不能洗脱下来，你怎么确定结合上去了？用酸、碱或者SDS之类的能洗下来吗？
结合真这么紧，到时候蛋白应该很纯啊。
50
also
.
【在 f*****s 的大作中提到】
: 最近用GE healthcare的glutathione sepharose 4B resin purify a couple of GST-
: tagged transcription factor DNA binding domains. The protein were bound to
: the beads with high efficiency. However, when I used glutathione buffer to
: elute the GST-protein off the beads, they can't be eluted. I tried 10 mM ,50
: mM and 100 mM glutathione. To make the buffer, I diluted 1 M Tris (pH 7.5
: at room temperature) to 50 mM, and added glutathione powder. There was also
: 2 mM DTT in the buffer. The elution was carried out at 4 degree where Tris
: should be at pH 8.0.
: I tried different resin and different glutathione, but none of them worked.
: So the problem must be in the buffer conditions.
结合真这么紧，到时候蛋白应该很纯啊。
50
also
.
【在 f*****s 的大作中提到】
: 最近用GE healthcare的glutathione sepharose 4B resin purify a couple of GST-
: tagged transcription factor DNA binding domains. The protein were bound to
: the beads with high efficiency. However, when I used glutathione buffer to
: elute the GST-protein off the beads, they can't be eluted. I tried 10 mM ,50
: mM and 100 mM glutathione. To make the buffer, I diluted 1 M Tris (pH 7.5
: at room temperature) to 50 mM, and added glutathione powder. There was also
: 2 mM DTT in the buffer. The elution was carried out at 4 degree where Tris
: should be at pH 8.0.
: I tried different resin and different glutathione, but none of them worked.
: So the problem must be in the buffer conditions.
a*e
6 楼
Chromcast能看中文节目吗？ 中国那些视频网站或支援Chromcast?
【在 t**b 的大作中提到】
: 出来的时候详细看了一下介绍，和chromecast是一类的东西。
: 除非比chromecast还便宜很多，否则应该什么太大市场。
【在 t**b 的大作中提到】
: 出来的时候详细看了一下介绍，和chromecast是一类的东西。
: 除非比chromecast还便宜很多，否则应该什么太大市场。
I*y
7 楼
p
50
also
.
the buffer pH to 8.0 after dissolving it in Tris.t be in the buffer
conditions.
【在 f*****s 的大作中提到】
: 最近用GE healthcare的glutathione sepharose 4B resin purify a couple of GST-
: tagged transcription factor DNA binding domains. The protein were bound to
: the beads with high efficiency. However, when I used glutathione buffer to
: elute the GST-protein off the beads, they can't be eluted. I tried 10 mM ,50
: mM and 100 mM glutathione. To make the buffer, I diluted 1 M Tris (pH 7.5
: at room temperature) to 50 mM, and added glutathione powder. There was also
: 2 mM DTT in the buffer. The elution was carried out at 4 degree where Tris
: should be at pH 8.0.
: I tried different resin and different glutathione, but none of them worked.
: So the problem must be in the buffer conditions.
50
also
.
the buffer pH to 8.0 after dissolving it in Tris.t be in the buffer
conditions.
【在 f*****s 的大作中提到】
: 最近用GE healthcare的glutathione sepharose 4B resin purify a couple of GST-
: tagged transcription factor DNA binding domains. The protein were bound to
: the beads with high efficiency. However, when I used glutathione buffer to
: elute the GST-protein off the beads, they can't be eluted. I tried 10 mM ,50
: mM and 100 mM glutathione. To make the buffer, I diluted 1 M Tris (pH 7.5
: at room temperature) to 50 mM, and added glutathione powder. There was also
: 2 mM DTT in the buffer. The elution was carried out at 4 degree where Tris
: should be at pH 8.0.
: I tried different resin and different glutathione, but none of them worked.
: So the problem must be in the buffer conditions.
t*b
8 楼
只要是chrome浏览器里能看的，chromecast就能'投影'到电视上。
因为是'投影'（技术上是把图像从计算机用无线网络传到chromecast设备，再在电视上
显示），播放还是用的计算机。大多数时候还是不如机顶盒方便，家有老人的话更是根
本用不了。而且诸如roku的机顶盒打折的时候$40, chromecast类的设备启示没什么优
势。
只有机顶盒上找不到节目，我才有时用一下chromecast，但很多这种时候也就直接用
laptop看了。
【在 a********e 的大作中提到】
: Chromcast能看中文节目吗？ 中国那些视频网站或支援Chromcast?
因为是'投影'（技术上是把图像从计算机用无线网络传到chromecast设备，再在电视上
显示），播放还是用的计算机。大多数时候还是不如机顶盒方便，家有老人的话更是根
本用不了。而且诸如roku的机顶盒打折的时候$40, chromecast类的设备启示没什么优
势。
只有机顶盒上找不到节目，我才有时用一下chromecast，但很多这种时候也就直接用
laptop看了。
【在 a********e 的大作中提到】
: Chromcast能看中文节目吗？ 中国那些视频网站或支援Chromcast?
s*y
9 楼
首先，先直接把resin 用2x SDS loading buffer煮一下直接上胶跑，
看看上面到底有没有蛋白.
第二，把resin 转移到小管里面去，加入elution buffer之后摇上10分钟，
然后再离心取上清。
50
also
.
【在 f*****s 的大作中提到】
: 最近用GE healthcare的glutathione sepharose 4B resin purify a couple of GST-
: tagged transcription factor DNA binding domains. The protein were bound to
: the beads with high efficiency. However, when I used glutathione buffer to
: elute the GST-protein off the beads, they can't be eluted. I tried 10 mM ,50
: mM and 100 mM glutathione. To make the buffer, I diluted 1 M Tris (pH 7.5
: at room temperature) to 50 mM, and added glutathione powder. There was also
: 2 mM DTT in the buffer. The elution was carried out at 4 degree where Tris
: should be at pH 8.0.
: I tried different resin and different glutathione, but none of them worked.
: So the problem must be in the buffer conditions.
看看上面到底有没有蛋白.
第二，把resin 转移到小管里面去，加入elution buffer之后摇上10分钟，
然后再离心取上清。
50
also
.
【在 f*****s 的大作中提到】
: 最近用GE healthcare的glutathione sepharose 4B resin purify a couple of GST-
: tagged transcription factor DNA binding domains. The protein were bound to
: the beads with high efficiency. However, when I used glutathione buffer to
: elute the GST-protein off the beads, they can't be eluted. I tried 10 mM ,50
: mM and 100 mM glutathione. To make the buffer, I diluted 1 M Tris (pH 7.5
: at room temperature) to 50 mM, and added glutathione powder. There was also
: 2 mM DTT in the buffer. The elution was carried out at 4 degree where Tris
: should be at pH 8.0.
: I tried different resin and different glutathione, but none of them worked.
: So the problem must be in the buffer conditions.
g*1
10 楼
Check the pH. Glutathione can drop pH to quite low. And bound protein can
not be eluted.
not be eluted.
f*s
11 楼
I've run SDS-PAGE so many times. The protein is clearly still on the beads.
I'll check the pH today. I just didn't think 10 mM glutathione can change
the pH of 50 mM Tris significantly.
I'll check the pH today. I just didn't think 10 mM glutathione can change
the pH of 50 mM Tris significantly.
s*e
12 楼
is there protease cutting site between ur proten and GST? if yes u can try
cut it from GST w/o elution.
cut it from GST w/o elution.
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