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Learn about Coomassie staining benefits and limitations, Western blotting techniques, Mass Spectrometry soft ionization, and separation techniques using Quadrupole Flight Tube. Discover how Coomassie interacts with proteins and its role in identifying purity. Analyze samples with MALDI-TOF MS for accurate results.
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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 Western Blot Coomassie Stained Benefits Limitations
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
MALDI Matrix α-cyano-4-hydroxycinnamic acid (CCA) 2,6-dihydroxyacetophenone (DHAP) SinapicAcid (SA)
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
Ideal Pairs ESI-QMS MALDI-TOF MS
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)