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Biochemistry Lab

Biochemistry Lab. Protein Purity Assessment and Identification. Coomassie Staining. How does Coomassie interact with proteins?. Coomassie Staining. Benefits. Limitations. Western Blotting. Transfer – Block Apply Primary Antibody Apply Secondary Antibody. Western Blots.

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Biochemistry Lab

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  1. Biochemistry Lab Protein Purity Assessment and Identification

  2. Coomassie Staining How does Coomassie interact with proteins?

  3. Coomassie Staining Benefits Limitations

  4. Western Blotting Transfer – Block Apply Primary Antibody Apply Secondary Antibody

  5. Western Blots Western Blot Coomassie Stained Benefits Limitations

  6. Mass Spectrometry

  7. Soft Ionization Techniques Matrix Assisted Laser Desorption Ionization (MALDI) Electrospray Ionization (ESI) • Aqueous sample introduced to metal capillary • High voltage (2000-4000 V) applied • Released to vacuum • Desolvation of aerosol leaving highly charged ions • Aqueous sample is cocrystallized on a metal surface with a Matrix • Intense Laser beam is directed toward sample/matrix mixture - desorption • Matrix absorbs the energy and is ionized • Some of the charge is transferred to the analyte

  8. MALDI Matrix α-cyano-4-hydroxycinnamic acid (CCA) 2,6-dihydroxyacetophenone (DHAP) SinapicAcid (SA)

  9. Separation Techniques Quadrupole Flight Tube • Four rods are arranged opposite each other and connected electronically • Voltage applied to each rod is carefully regulated • The trajectory of a charged particle is influenced by the electric field • Molecules separate by the time it takes for them to travel from the ion source to the detector • Resolution is dependent on tube length (limits resolving power) • Reflectron enhances the resolution

  10. Ideal Pairs ESI-QMS MALDI-TOF MS

  11. Other Applications of MS

  12. In lab this week • Run and stain (coomassie) the gels you prepared last week • Use Fractions 1-9 and a MW ladder • MALDI-ToF MS of your fractions SinapicAcid (SA)

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