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Sucrose Gradient Analysis of Proteins   

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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, Molecular weight of the protein, S value

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.
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Zhu, H. (2012). Sucrose Gradient Analysis of Proteins. Bio-101: e194. DOI: 10.21769/BioProtoc.194.
Q&A
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If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

10% is 10 g/100 ml
40% is 40 g/100 ml
2/19/2013 11:03:53 AM Reply
Hui Zhu
Stanford University

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.

2/28/2013 5:48:25 PM Reply


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