Methods for Total Protein Flashcards
Enumerate the Methods for Total Protein
- Kjeldahl Method
- Biuret Method
- Folin-Ciocalteu (Lowry Method)
- UV Absorption Method
- Refractometry
- Turbidimetry and Nephelometry
- Coomasie Brilliant Blue Dye
- Ninhydrin
- Serum Protein Electrophoresis (SPE)
- Classical method for protein quantitation
- Reference method but not routinely used because it is very time consuming and only uses an assumption
- Not accurate
Kjeldahl Method
T/F. In Kjeldahl Method, Measures the amount of nitrogen in specimen (Protein = CHON).
True
- Assumes the nitrogen content of protein is 16% (although, it varies between 15.1%-16.8%)
Kjeldahl Method
In Kjeldahl Method, 1g of nitrogen is equal to
6.54% of protein
3 steps: In Kjeldahl Method
Digestion, Distillation, Titration
- A colorimetric, non-enzymatic method
- Most widely used method for total protein determination
Biuret Method
Biuret Method End product:
Violet-colored
Biuret Method Absorbance of color is read at
540nm
Measures peptide bonds present in protein forms a bond with cupric ions forming a violet-colored chelate
Biuret Method Principle
T/F. In Biuret Method, More peptide bonds = Dark color = More protein
True
Biuret Method Interference:
Lipemic sample
Enumerate the Biuret reagent
o Alkaline CuSo4
o NaK tartrate (Rochelle salt) – to prevent precipitation of copper
o NaOH
o KI – stabilizer
Has the highest analytical sensitivity and can measure even the smallest amount of protein
Folin-Ciocalteu (Lowry Method)
Oxidation of phenolic compounds such as tyrosine, tryptophan and histidine to give a deep blue color (measures spectrophotometrically)
Folin-Ciocalteu (Lowry Method) Principle
Folin-Ciocalteu (Lowry Method) Main reagent:
Phosphotungstic-Molybdic acid or phenol reagent
T/F. Phosphotungstic-Molybdic acid or phenol reagent, can oxidized phenolic compounds
True
Color enhancer use in Folin-Ciocalteu (Lowry Method)
Biuret reagent
The absorbance of proteins at 210nm is due to the absorbance of the peptide bonds at specific wavelength
UV Absorption Method Principle
T/F. In UV Absorption Method, Most proteins has 210nm absorption
True
Absorption at 280nm:
Aromatic AA (Tryptophan, tyrosine and phenylalanine)
- Based on measurement of refractive index of serum total proteins
- Based on how protein bend light which is directly proportional (Increase changes = More protein)
Refractometry
Measurement depends on formation of a uniform fine precipitate which scatter incident light in suspension (nephelometry) or block light (turbidimetry)
Turbidimetry and Nephelometry
Used for detection of proteins as little as 1ug
Coomasie Brilliant Blue Dye
