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
Develops violet color by reacting with primary amines widely used for detection of peptides and amino acids after chromatography
Ninhydrin
it is a type of separation technique
Serum Protein Electrophoresis (SPE)
Migration of charged particles in an electric field
Serum Protein Electrophoresis (SPE) Principle
Negative terminus →
cathode
Positive terminus →
anode
T/F. In (SPE), Isoelectric property of proteins (they go to anode)
True
either positive or negative depending on pH condition
Amphoteric
(SPE), Increase pH (basic) =
Negative
(SPE), Decrease pH (acid) =
Positive
T/F. In SPE, No charge at isoelectric point (Net charge: 0)
True
The acidic and basic amino acids content of proteins determines its net charge
SPE
SPE Buffer:
Barbital (Veronal) pH 8.6
