Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
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Small Scale Sucrose Density Gradient Centrifugation for Protein and/or Cellular Organelle Isolation |
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| Contributor: |
The Laboratory of Andrew Murray at Harvard University | ||||||||||||
| Overview |
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| Sucrose gradients are frequently used for separating cell organelles from crude cellular extracts. Additionally, sucrose gradients can be used as the first "crude" separation of cytosolic and/or nuclear proteins. This protocol is a general description of the sucrose gradient method. Sucrose concentrations should be determined for specific applications. | |||||||||||||
| Procedure |
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1. Prepare three 50 ml tubes labeled as follows: 13.75%, 22.5%, 31.25%. 2. Add 10 ml of 5% Sucrose and 10 ml of 40% Sucrose to the tube labeled 22.5%. 3. Add 10 ml of 5% Sucrose and 10 ml of 22.5% Sucrose to the tube labeled 13.75%. 4. Add 10 ml of 40% Sucrose and 10 ml of 22.5% Sucrose to the tube labeled 31.25%. 5. Add the following Sucrose solutions sequentially in Beckman SW55 Ti rotor tubes: Use a large bore pipette tip (cut off approximately 2 mm from the tip of a 1 ml pipette tip) and "layer" the solutions very slowly into the tube (see Hint #2) 950 μl of 40% Sucrose 950 μl of 31.25% Sucrose 950 μl of 22.5% Sucrose 950 μl of 13.75% Sucrose 950 μl of 5% Sucrose 6. Gently keep the tube of Sucrose solution layers at 4°C for at least 12 to 16 hr (i.e., overnight). A linear gradient will form. 7. Layer the crude cellular extract on top of the Sucrose gradient solution (approximately 100 μl and see Hint #3). 8. Insert the tube into a pre-chilled SW55 Ti rotor at 4°C. Balance the rotor with another tube of equal mass and centrifuge the Sucrose gradient at 4°C for 4 hr at 50,000 rpm (approximately 237,000 X g). 9. After centrifugation, prepare sixteen microcentrifuge tubes on ice. Remove the centrifuge tube from the rotor and place on ice. 10. Carefully transfer 300 μl of fractions from the top of the gradient to the microcentrifuge tubes with large-bore pipette tips. 11. Assay the fractions by SDS-PAGE, and/or concentrate the protein fractions with TCA precipitation (see Protocol ID#455 and Protocol ID#1143). |
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| Solutions |
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| BioReagents and Chemicals |
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Sucrose |
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| Protocol Hints |
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1. Typically, the buffer will be the same composition as the extract to be fractionated by centrifugation. The presence of Sucrose or Glycerol in the starting extract will also affect the migration of proteins. 2. It is useful to prop the centrifuge tube in a bucket of ice and tilt the tube to approximately 30° to assist in the gentle layering of each Sucrose solution on top of the previous solution. If the layering is not gentle enough, the fractionation will be smeary. While layering, notice the barely visible layers of solution. 3. Be careful not to overload the gradient with too much protein. This requires empirical determination for the protein of interest and its migration in Sucrose. |
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