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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



western blotting
Western Blotting

Transfer – Block

Apply Primary Antibody

Apply Secondary Antibody

western blots
Western Blots

Western Blot

Coomassie Stained



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


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



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)