MEASUREMENT OF PROTEINS Flashcards
▪ determination of protein nitrogen derived from constituent amino acids
KJELDAHL METHOD
▪ Nitrogen in pff is converted to ammonia using
H2SO4
The nitrogen in ammonia may be measured using:
Nesslerization
Berthelot reaction
Titration method
▪ Copper binds to the peptide bond structure of the protein, forming a purple-colored chromogen
BIURET METHOD
▪ Tyrosine & tryptophan in proteins reduce PT-PMA reagent (folin-ciocalteu) to give a blue color
FOLIN-CIOCALTEU METHOD
▪ Detects proteins in concentrations as low as 10 – 60 µg/mL
FOLIN-CIOCALTEU METHOD
▪ Widely used in research to measure tissue & enzyme proteins
FOLIN-CIOCALTEU METHOD
▪ Chiefly the result of the high electron density of aromatic rings of tyrosine & tryptophan in solution (pH 8)
Absorption of UV light at 280 nm
▪ Based on the ability of proteins to bind certain dyes
Dye-Binding Methods
▪ Bromcresol Green Method (BCG) for Albumin
Dye-Binding Methods
▪ Used to stain protein bands after electrophoresis (e.g. Coomassie Brilliant Blue)
Dye-Binding Methods
▪ direction of migration of proteins in an electrical field determined by surface charge & MW of protein
PROTEIN ELECTROPHORESIS
Urine protein electrophoresis exactly same as serum except it must be concentrated before application
PROTEIN ELECTROPHORESIS
PROTEIN ELECTROPHORESIS
Support media include
cellulose acetate, agarose gel and starch gel
PROTEIN ELECTROPHORESIS
Stains:
amido black, ponceau S and coomassie brilliant blue
