BIOC312 Lecture Notes - Lecture 1: Particle Size, Antigen, Fragment Antigen-Binding

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22 Jul 2016
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Biochemistry Part IILecture 1 and 2; Protein Purification Protein purification;
Disruption of cells and tissuesDifferentials centrifugation to isolate cellular
cellular organelleSeparation of proteins on the basis of; solubility, size,
charge, hydrophobiccity, affinity for ligands
Separation of organelles from animal cells by differential centrifugation
--Number of times gravity for the time period
Centrifugation of homogenate at 500 G for 10 minutes gives
nuclear fraction (pellet)
Centrifugation of homogenate at 10,000 G for 20 minutes gives a
mitochondrial fraction (pellet)
Centrifugation of homogenate at 100,000 G for for 60 minutes
gives a microsomal fraction (pellet)
Resulting supernatant contains cytosol (Soluble proteins)
Centrifugal force increases according to the square of the angular velocity
Where r is radius in meters, is angular velocity =2 (rpm)/60 ω π
Crude separation of proteins on the basis of solubility
Proteins can be precipitated by adding competing solutes such as
ammonium sulphate and polyethylene glycol.
Precipitates are removed by centrifugation.
Competing solutes remove hydration shells from proteins so proteins
accumulate.
Different proteins cling to hydration shells with different strengths and
basis of size and charge
Ideal competing solutes are;
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Very water soluble(up to 50% weight/volume)
Relatively non denaturing to proteins in general
Easier to remove the protein afterwards
Not too viscous or dense
Cheap and pure
Crude separation of proteins on the basis of size
In dialysis (small molecules pass through tiny pores in a plastic
bag, large particles cannot pass through)
SEC, or size exclusion chromatography. Here, large molecules
cannot enter polymetric beads in the column. Small molecules
can enter the beads, and therefore pass through a ..... Small
molecules pass through slower than large molecules.
Separation on the basis of charge; ion exchange chromatography
Positively charged proteins bind to the negatively charged beads,
allowing negatively charged proteins to flow through
Elution of pound protein from the column can be bought about by
competition with increasing concentrations of NaCl
Affinity chromatography; the protein is specifically bound to the column and
includes a later
Immunoaffinity chromatography
Immobilised ligand chromatography
Addition of affinity “tags” to recombinant proteins (Poly-histidine tag
at N- or C- terminus of the protein) is widely used
Lectin based affinity chromatography (Bonds glycosylated proteins). Elution
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