(3) METHODS FOR TP Flashcards
- Classical method for protein quantitation
- Reference method but not routinely used:
o Time-consuming
o Only an assumption so it is an inaccurate method
Kjeldahl method
Principle: Measures the amount of Nitrogen in spx
Kjeldahl method
3 steps of Kjeldahl method
Digestion → Distillation → Titration
- Assumes that nitrogen content of protein is only 16% when it actually varies between 15.1-16.8%
- 1g of nitrogen = 6.54g of protein
Kjeldahl method
- Most widely used method for TP determination
- Colorimetric/Non-enzymatic method
Biuret method
Absorbance of color is read @ 540nm
(deep purple/violet)
Biuret method
Principle: Based on # of peptide bonds present in protein forms a bond w/ cupric ion forming a violet-colored chelate.
Biuret method
o The darker the color, the more protein is present.
o How? Cu in alkaline CuSO4 will bind to peptide bonds present in protein forming a peptide-copper complex giving a violet-colored product.
Biuret method Interference:
Lipemic sample
Biuret method Reagents
o NaK tartrate (Rochelle salt): prevents ppt of Cu
o NaOH
o Alkaline CuSO4
o KI: acts as stabilizer
- Principle: Oxidation of phenolic AA compounds such as tyrosine, tryptophan, & histidine.
- Has the highest analytical sensitivity (can detect very small amounts of protein)
Folin-Ciocalteu (LOWRY) method
Folin-Ciocalteu method main reagent:
Phosphotungstic-molybdic acid or phenol rgt
o Can oxidize phenolic AA causing reduction of phosphotungstic-molybdic acid → deep blue color measured spectrophotometrically.
Folin-Ciocalteu method color enhancer:
Biuret rgt
- Principle: The absorbance of proteins at 210nm is due to the absorbance of the peptide bonds at specific wavelength.
UV ABSORPTION method
UV absorption @ 280nm:
Aromatic AA such as tryptophan, tyrosine, phenylalanine
- Based on measurement of refractive Index of serum TP
- Based on how protein bend light.
- Changes is proportional to conc. of proteins in sample.
Refractometry method
