欢迎您, 登录 | 注册

首页 | English

X
加载中

The sedimentation rate of a protein in a linear sucrose gradient can be used to determine its S value when compared to proteins of known S values which are run in a parallel gradient. This can be used to estimate an approximate molecular weight of the protein and test the potential interaction of two proteins if they peak in the same gradient range.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

[Bio101] Sucrose Gradient Analysis
[Bio101] 蔗糖密度梯度分离

生物化学 > 蛋白质 > 定量
作者: Hui Zhu
Hui ZhuAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: huizhu@stanford.edu
Bio-protocol author page: a32
2/5/2012, 14898 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.194

[Abstract] The sedimentation rate of a protein in a linear sucrose gradient can be used to determine its S value when compared to proteins of known S values which are run in a parallel gradient. This can be used to estimate an approximate molecular weight of the protein and test the potential interaction of two proteins if they peak in the same gradient range.
Keywords: Sucrose gradient(蔗糖梯度离心), Molecular weight of the protein(蛋白质的分子量), S value(S 值)

[Abstract] 蛋白在蔗糖梯度中的沉降率可以用于与已经知道的S值与其在同梯度中蛋白相比来确定其S值。从而可用于计算蛋白的大约的分子量,也可用于鉴定两个蛋白的相互作用,如果他们在相同梯度出峰。

Materials and Reagents

  1. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  2. Gelatin (Sigma-Aldrich, catalog number: G9136 )
  3. Aldolase (Bio-Rad Laboratories)
  4. Thyroglobulin (Bio-Rad Laboratories)
  5. Sucrose solutions (see Recipes)

Equipment

  1. Centrifuges (Beckman Falcon, TLS-55 )
  2. Gradient mixer (Sigma-Aldrich)
  3. Stirring bar

Procedure

  1. Make 10% and 40% sucrose solutions in the same buffer used for the protein sample.
  2. Coat the tubes with 1% gelatin.
  3. Fill the tube with 1% gelatin solution.
  4. Pour the gelatin out and wash several times with ddH2O.
  5. Make a 10-40% linear sucrose gradient.
    Note: There are two chambers in a gradient mixer, a reservoir and a mixing chamber, with an interconnecting valve. A second valve regulates the output flow from the mixing chamber. A magnetic stirring bar can be placed in the mixing chamber to maintain a constant gradient. All of the mixers have a flat base to enable them to be placed on a magnetic stirrer.
  6. Put the gradient mixer on top of a stir plate so that the gradient maker is level. Put a small stir bar in mixing chamber.
  7. With all valves closed, fill each chamber with 1/2 the total amount of desired gradient [i.e. 1.1 ml for a 2.2 ml gradient ( TLS-55 )]. Use 40% sucrose in mixing chamber and 10% sucrose in reservoir chamber.
  8. Turn on the stir plate to a setting so it is mixing gently.
  9. Place the output tube in the centrifuge tube so that it is just touching the side near the top.
  10. Open both valves at the same time. The 40% sucrose should start filling the centrifuge tube and the 10% sucrose should start mixing with the 40% sucrose.
  11. Close both valves when the gradient nears the top of the centrifuge tube. Be sure to leave enough room to layer the sample on top of the gradient.
  12. Put the gradients and rotor in the cold room for between 2-48 h. Make sure the tubes are balanced and then layer the sample on top. The samples should be of a small volume (e.g. 100 μl for a 2.2 ml gradient).
  13. Spin at 55,000 rpm for 2.5 h.
  14. Collect fractions to analyze by western blotting. Aldolase (158 kDa, 7S) and thyroglobulin (690 kDa, 19S) were used as size markers and analyzed in parallel. Ideally the protein of interest should be in the middle of the gradient.

Recipes

  1. 10% sucrose solutions
    10 mg sucrose in 100 ml ddH2O
  2. 40% sucrose solutions
    40 mg sucrose in 100 ml ddH2O

Acknowledgments

This protocol was developed in the laboratory of Dr. Guowei Fang (Department of Biology, Stanford University, Stanford, CA, USA). This work was supported by a Burroughs-Wellcome Career Award in Biomedical Research (G.F.) and by grants from National Institutes of Health (GM062852 to G.F.).

References

  1. Martin, R. G. and Ames, B. N. (1961). A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem 236: 1372-1379.
  2. Zhu, H., Coppinger, J. A., Jang, C. Y., Yates, J. R., 3rd and Fang, G. (2008). FAM29A promotes microtubule amplification via recruitment of the NEDD1-gamma-tubulin complex to the mitotic spindle. J Cell Biol 183(5): 835-848.

材料和试剂

          

1.        蔗糖(Sigma Catalog No. S0389)

2.        明胶(Sigma Catalog No. G9136)

 

仪器

 

1.        离心机(Beckman Falcon, TLS-55)

2.        梯度混合器 (Sigma)

 

步骤

 

1.         用与蛋白样品缓冲液相同的缓冲液制作10% 40% 的蔗糖溶液。

2.         用的1%明胶包被管子,用1%的明胶溶液加满管子,倒出凝胶溶液并用ddH2O.洗涤。

3.         制作 10-40% 蔗糖梯度

注意:梯度混合器有两个室,储存室和混合室,并用一个互联的膜隔开。用另一个膜来调控从混合器中流出。在混合室中的磁力搅拌器可以帮助保持一个不变的梯度所有的混合器都有一个平面用于放在磁力搅拌器上。

4.         将梯度混合器放在磁力搅拌器上面,使其水平。将一个小的搅拌子放入混合室中。

5.         使所有瓣膜关闭,用1/2想要的梯度量加入每个室中, (i.e. 1.1 ml 对于 2.2 ml 梯度(TLS-55)).  40% 在混合室, 10%蔗糖在储存室。

6.         打开磁力搅拌器使得它轻轻的搅拌。

7.         将输出管放到一个离心管中,使起刚刚碰到接近顶部的边。

8.         同时打开两个膜, 40% 蔗糖溶液开始进入离心管, 10%蔗糖开始与40%蔗糖混合。当梯度接近离心管的顶部关闭两个瓣膜。确定有足够的地方放顶部的梯度样品。.

9.         把梯度的转子放在冷室中2-48 hrs. 确定管子平衡并且样品在顶部,样品只占很少的体积(e.g. 100 μl对于2.2 ml 梯度溶液)

10.     55,000 rpm离心2..5 小时。

11.     收集组分并且用Western blotting检测。醛缩酶(158 kD, 7S) 和甲状腺球蛋白(690 kD, 19S) (Bio-Rad Laboratories) 并用于marker被用于一起检测,我鉴定的感兴趣的蛋白在梯度溶液的中间。

 

配置方法

 

1.      10% 蔗糖溶液:10 mg 蔗糖溶于100 ml ddH2O

2.      40%蔗糖溶液:40 mg蔗糖溶于100 ml ddH2O

 

摘要

 

1.       Martin R.G., Ames B.N. (1961). A method for determining the sedimentation behavior of enzymes: Application to protein mixtures. Journal of Biological Chemistry 236: 1372-9. 

2.       Zhu H., Coppinger J.A., Jang C.Y., Yates J.R., 3rd, Fang G. (2008). FAM29A promotes microtubule amplification via recruitment of the NEDD1-gamma-tubulin complex to the mitotic spindle. Journal of Cell Biology 183(5): 835-48. 

 

 

English
中文翻译

免责声明

为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。

X


How to cite this protocol: Zhu, H. (2012). Sucrose Gradient Analysis. Bio-protocol Bio101: e194. DOI: 10.21769/BioProtoc.194; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
2/19/2013 11:03:53 AM  

10% is 10 g/100 ml
40% is 40 g/100 ml

2/28/2013 5:48:25 PM  

Hui Zhu (Author)
Department of Genetics, Stanford University, USA

Yes, You are right:
10% sucrose solutions: 10 g sucrose in 100 ml ddH2O
40% sucrose solutions: 40 g sucrose in 100 ml ddH2O

Thanks.

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register