purpose of sucrose in buffer - organelle separation - (Oct/07/2007 )
Dear people,
again questions 
.
1. in many papers there is a "protocol" for preparation of (plasma) membrane, mitochondrial, nuclear fraction.
They use, for example, 20mM HEPES, pH=7.4, protease inhibitors, and 0.25M sucrose, then after homogenization, centrifugation at 1000xg (nuclear pellet), 10000xg (mitochondrial pellet), 100 000xg (membrane pellet) and cytosol in supernatant.
What is the purpose of sucrose in the buffer?
What would happen if sucrose would not be there (organelle would be broken)?
2. Triton X-100 soluble and insoluble fraction. Usually (in many papers) it is used 1% of Triton X-100 (but I have seen also even 2%).
Why only 1% of Triton X-100? Can I use 2% or it is too much?
3. Is it problem of presence of 0.25M sucrose in a sample (at the protein purification) for anion exchange, reverse phase chromatography?
Again thanks for help,
vic
again questions
.1. in many papers there is a "protocol" for preparation of (plasma) membrane, mitochondrial, nuclear fraction.
They use, for example, 20mM HEPES, pH=7.4, protease inhibitors, and 0.25M sucrose, then after homogenization, centrifugation at 1000xg (nuclear pellet), 10000xg (mitochondrial pellet), 100 000xg (membrane pellet) and cytosol in supernatant.
What is the purpose of sucrose in the buffer?
What would happen if sucrose would not be there (organelle would be broken)?
2. Triton X-100 soluble and insoluble fraction. Usually (in many papers) it is used 1% of Triton X-100 (but I have seen also even 2%).
Why only 1% of Triton X-100? Can I use 2% or it is too much?
3. Is it problem of presence of 0.25M sucrose in a sample (at the protein purification) for anion exchange, reverse phase chromatography?
Again thanks for help,
vic
1. Sucrose acts as a cushion, and give you better separation of cell fractionation, less contamination between these fraction. In some protocol, it will also call for overlaying the cell lysate over sucrose cushion (at higher conc. than 0.25M) and then centrifuge for the same purpose.
2. you don't want to use too much detergent because it could affect your protein folding, and/or interaction. So you always start with milder condition (less amount and with mild detergent) first and only go up if that condition does not serve your purpose. If 1% TX-100 is enough to solubilize your protein, what is the use of 2%?
1) to expand on almasy's response, the sucrose increases the density of the medium. this way you can separate the organelles by differential centrifugation, as you describe. if the sucrose wasn't there then you would pellet all three fractions in the same spin.
3) the sucrose won't have an effect on ion exchange other than to increase back pressure while the sample is being loaded.
i'm not sure if it will have an effect on reversed phase but it may be worth trying.