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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
Biochemistry Lab

Protein Purity Assessment and Identification

coomassie staining
Coomassie Staining

How does Coomassie interact with proteins?

coomassie staining1
Coomassie Staining

Benefits

Limitations

western blotting
Western Blotting

Transfer – Block

Apply Primary Antibody

Apply Secondary Antibody

western blots
Western Blots

Western Blot

Coomassie Stained

Benefits

Limitations

soft ionization techniques
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
MALDI Matrix

α-cyano-4-hydroxycinnamic acid (CCA)

2,6-dihydroxyacetophenone (DHAP)

SinapicAcid (SA)

separation techniques
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
Ideal Pairs

ESI-QMS

MALDI-TOF MS

in lab this week
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)