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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.
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Biochemistry Lab
Protein Purity Assessment and Identification
Coomassie Staining
How does Coomassie interact with proteins?
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
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
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)
