ProtPurification

Beads in column vo volume outside beads vi volume

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Unformatted text preview: lumn: Vo = volume outside beads; Vi = volume outside+inside beads Excluded (large) Partially included Included (salt) [Protein] Vo Vi Volume of eluate Other size techniques: zonal untracentrifugation, ultrafiltration Charge Electrophoresis: F = Eq/d – 6πrηv; v(velocity) = E(voltage) q(charge) / 6πr(x-section) η(viscosity) d(electrode separation) negative electrode starch gel, stabilized liquid, etc. less negative protein (surface charge density) more negative protein positive electrode SDS (sodium dodecylsulfate) - PAGE (polyacrylamide gel eletrophoresis) (size again)/ SDS: charged detergent: denatures protein and gives uniform surface charge density acrylamide gel: cross-links produce resistance to protein movement negative electrode larger protein smaller protein positive electrode Isoelectric electric focusing (separate by pH at which proteins have zero charge) use “ampholytes” to stabilize pH gradient in electrophoresis gel protein moves to pI (isoelectric point, zero charge) negative electrode pH 3 high pI (e.g. 10) 10 larger low pI (e.g. 4) positive electrode smaller Note 2-D gels combine isoelectric focusing with SDS-electrophoresis Biological specificity: affinity chromatography 1. 2. 3. 4. Attach “ligand” to insoluble bead Combine protein mixture with beads: enzyme binds to ligand Load column; wash column Elute enzyme with substrate or change in pH (to reduce binding) Protein (solid) Sp. ac. (dashed) load wash elute What do you use for ligand: substrate analog; dyes simulate enzyme cofactors (often planar, aromatic)...
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