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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.

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[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, 16630 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-Aldrich,目录号:SO389)
  2. 明胶(Sigma-Aldrich,目录号:G9136)
  3. 醛缩酶(Bio-Rad Laboratories)
  4. 甲状腺球蛋白(Bio-Rad Laboratories)
  5. 蔗糖溶液(见配方)

设备

  1. 离心机(Beckman Falcon,TLS-55)
  2. 梯度混合器(Sigma-Aldrich)
  3. 搅拌棒

程序

  1. 在用于蛋白质样品的相同缓冲液中制备10%和40%蔗糖溶液
  2. 用1%明胶包被试管。
  3. 用1%明胶溶液填充试管。
  4. 倒出明胶,并用ddH 2 O洗涤数次。
  5. 制成10-40%线性蔗糖梯度。
    注意:在梯度混合器中有两个室,一个储存器和一个混合室,具有一个互连阀。 第二阀调节来自混合室的输出流量。 可以将磁性搅拌棒放置在混合室中以保持恒定的梯度。 所有的搅拌机都有一个平底,可以放在磁力搅拌器上。
  6. 将梯度搅拌器放在搅拌板的顶部,使梯度仪水平。 将一个小搅拌棒放在混合室中。
  7. 在所有阀关闭的情况下,用1/2的所需梯度的总量(即对于2.2ml梯度(TLS-55)为1.1ml)填充每个室。在混合室中使用40%蔗糖,在储存室中使用10%蔗糖
  8. 打开搅拌盘到一个设置,使其轻轻混合。
  9. 将输出管放在离心管中,使其刚好接触顶部附近的一侧。
  10. 同时打开两个阀门。 40%蔗糖应开始填充离心管,10%蔗糖应开始与40%蔗糖混合。
  11. 当梯度接近离心管顶部时关闭两个阀门。一定要留出足够的空间将样品层叠在梯度顶部
  12. 将梯度和转子放在冷室中2-48小时。确保管道平衡,然后将样品在顶部。样品应具有小体积(例如,对于2.2ml梯度,100μl)。
  13. 以55,000rpm旋转2.5小时。
  14. 收集级分,通过western印迹分析。 醛缩酶(158kDa,7S)和甲状腺球蛋白(690kDa,19S)用作大小标记物,并平行分析。 理想地,感兴趣的蛋白质应该在梯度的中间

食谱

  1. 10%蔗糖溶液 10mg蔗糖在100ml ddH 2 O中的溶液
  2. 40%蔗糖溶液
    40mg蔗糖在100ml ddH 2 O中的溶液

致谢

这个协议是在国防部的实验室(斯坦福大学生物系,斯坦福,加利福尼亚州,美国)开发的。 这项工作得到了生物医学研究(G.F.)的Burroughs-Wellcome职业奖和国立卫生研究院(GM062852至G.F.)的资助。

参考文献

  1. Martin,R.G。和Ames,B.N。(1961)。 确定酶的沉降行为的方法:应用于蛋白质混合物。 > J Biol Chem 236:1372-1379
  2. Zhu,H.,Coppinger,J.A.,Jang,C.Y.,Yates,J.R.,3rdand Fang,G。(2008)。 FAM29A通过募集NEDD1-γ-微管蛋白复合物到有丝分裂纺锤体来促进微管扩增。 a> J Cell Biol 183(5):835-848
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How to cite this protocol: Zhu, H. (2012). Sucrose Gradient Analysis. Bio-protocol Bio101: e194. DOI: 10.21769/BioProtoc.194; Full Text



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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.

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