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Physical Methods to Characterize Proteins

Physical Methods to Characterize Proteins. Physical properties of key interest. Molecular weight. Oligomerization state. Structure. Interactors. Transport Processes. 1. Transport in an electric field ( electrophoresis ).

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Physical Methods to Characterize Proteins

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  1. Physical Methods to Characterize Proteins

  2. Physical properties of key interest Molecular weight Oligomerization state Structure Interactors

  3. Transport Processes 1. Transport in an electric field (electrophoresis) Requires a matrix (either agarose or polyacrylamide) to minimize convective effects due to heating and Brownian motion. Mobility is also affected by partioning with the stationary phase (matrix). Provides relative molecular weights determined by comparison to standards. 2. Transport in a gravitational field (centrifugation) Requires a centrifuge to generate the large gravitational forces required for protein transport. Provides an absolute molecular weight and informs on the oligomerization state for stable complexes. 3. Transport by partitioning between mobile and stationary phases (gel exclusion chromatography) Requires a suitably-sized partitioning matrix for the solid phase. Provides relative molecular weights determined by comparison to standards.

  4. Gel Electrophoresis u = A(q/f) Electrophoretic mobility (u) is proportional to its net charge (q), specifically the ratio of its net surface charge to accessible surface area, and inversely proportional to its frictional coefficient (f), a function of solvent viscosity and protein geometry. The constant of proportionality (A) is unique to each protein. Mobility can be measured to the cathode or anode depending on protein charge and pH. Free radicals are provided by ammonium persulfate. TEMED (tetramethylenediamine) is included to stabilize the free radicals resulting from decomposition of the ammonium persulfate. Ratio of acrylamide to bisacrylamide is held constant. Pore size is expressed as percent (w/v) acrylamide and designated %T.

  5. Disc (Discontinuous) Gel Electrophoresis Changes in electrophoretic mobility in the focusing zone Leading anion: Cl- Trailing ion: glycine

  6. SDS PAGE SDS: Sodium Dodecyl Sulfate (Lauryl sulfate) SDS binds protein with a constant ratio of 1.4 gm SDS/gm protein. Calibration plot for a series of molecular weight standards vs. %T

  7. Gel Filtration/Size Exclusion Chromatography Comparison of size resolution by gel filtration (left) and PAGE (right) Calibration plots for molecular weight standards resolved on different media Band spreading as a function of elution position

  8. Mass Spectrometry Ionization methods Matrix-assisted Laser desorption/ionization (MALDI) Basic mass spectrometer design Electrospray ionization (ESI) Detection methods Time of Flight (TOF)- Accuracy to 0.1% Quadrupole mass analyzers- Accuracy to 0.01% FT ion cyclotron- Accuracy to 0.001%

  9. Types of MS Data Mass determination of intact proteins Tandem mass spectrometry analysis by collision-induced dissociation (CID) Tandem mass spectrometry

  10. SILAC-Stable Isotope Labeling by Amino Acids

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