4-423-08ProteinAnal

4-423-08ProteinAnal - Pour through protein mixture Discard...

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20 kg fresh beef heart Cold 0.2 M sucrose pH 7.2 Hint: other tissue possibilities for this enzyme are bird or insect flight muscle…
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----------------- ----------- WHAT HAPPENS TO THE TISSUE WHEN IT IS HOMOGENIZED? WHY 0.2 M SUCROSE? WHY pH 7.2, AND COLD? Organelle
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WHY DIFFERENTIAL CENTRIFUGATION? NEXT, OSMOTICALLY LYSE MITOCHONDRIAL FRACTION… -------------------------------------> AMMONIUM SULFATE ADDED IN TWO STEPS: WHY? (citrate synthase) 1st salt addition Unwanted proteins 2nd salt addition Target protein(s) * pH 7.2 Why? Removal of excess ammonium sulfate salt Dialysis: Cold, overnight, several changes of buffer (solvent)
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Overview of chromatographic techniques Size-exclusion chromatography First protein fraction… What does this infer about citrate synthase?
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Why is UV absorption at 280 nm a good way to monitor the presence of protein? Cation exchange column, pH 7.2
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Unformatted text preview: Pour through protein mixture Discard initial flow-through Add higher pH wash Collect eluted protein fraction What does this infer about our protein? What amino acid residues are affected by the pH change?----------- -----------------------------------------pH 5.6 <--Isoelectric focusing gel (IEF gel) pI is a function of all the ionizable groups on a protein Gel separation techniques Why was I unconvinced of the purity of my IEF gel band? 2-D gels use pI, then mass, to separate proteins <--SDS polyacrylamide gel electrophoresis (SDS- PAGE) What does one band on the SDS PA gel (after IEF) tell me? Why do the SDS-PAGE after IEF? Denaturation, renaturaton (Or, drastic changes in pH, ionic strength, temperature usually lead to irreversible denaturation)...
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4-423-08ProteinAnal - Pour through protein mixture Discard...

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