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Protein Fractionation
Separating proteins based on physical or chemical properties
How were proteins fractionated the PF lab?
Acid Precipitation and Salting Out
Acid Precipitation
Separation based on pH.
Precipitate unwanted proteins by passing through their isoelectric point
Salting out
Separation based on ionic strength
Dependent on hydrophobicity - proteins with more hydrophobic regions will precipitate first
Done at 4°C → decrease risk of inactivation
What are common issues that could occur during protein fractionation, and how do they affect the results?
No or small pellet formation
Low absorbance readings in CB assay
Cloudy supernatant after centrifugation
Overlap between fractions
No clear trend in protein concentration across fractions
Why do we fractionate proteins?
To isolate proteins of interest based on specific chemical or physical properties
Increase accuracy for analysis such as electrophoresis
Reagents used in the PF lab
Tissue Extract
Protein Buffer
Acid Precipitation Buffer
Ammonium Sulfate Salt Solution
CB Protein Assay Reagent
Protein Standard
Aims of the PF Lab
Learn protein purification strategies
Lean protein fractionation principles
Protein fractionation by changing pH/salting out
PF Methods
Precipitate the proteins with an unwanted pH through acid precipitation
Precipitate the proteins that are more hydrophobic through salting out at 3 levels of ammonium sulfate concentration
Protein Fractionation principles learned
Isoelectric Precipitation
Salting Out