METHODS FOR TOTAL PROTEIN Flashcards
Classical method for protein quantitation
Kjeldahl Method
Reference method but not routinely used; TIME
CONSUMING
Kjeldahl Method
Kjeldahl Method
Measures the amount of __________ in specimen
Nitrogen
Kjeldahl Method
1 g of nitrogen = ?
6.54 g of proteins
Kjeldahl Method
3 STEPS:
a. DIGESTION
b. DISTILLATION
c. TITRATION
COMPONENTS OF THE PROTEIN:
CHON –
carbohydrate, oxygen and nitrogen
Measures protein based on the number of peptide
bonds present.
Biuret Method
Biuret Method
More peptide = ?
darker the color
The darker the color the more protein present in
the sample
Biuret Method
Biuret Method
The ______ present will bind to the peptide bonds
present in the protein forming
Copper
Biuret Method
peptide copper
complex = ?
violet
Most widely used method for total protein
determination
Biuret Method
Biuret Method
_______ because the end product has color
(“DEEP COLOR PURPLE”/VIOLET)
Colorimetric
Biuret Method
principle pr reagent do
not utilize enzyme
Non-enzymatic method
Biuret Method
Reagent consists:
a. Alkaline CuSO4
b. Sodium potassium tartrate (Rochelle
salt)
c. NaOH
d. Potassium iodide –
Biuret Method
Absorbance of color is read at ?
540 nm
Biuret Method
present in protein forms a
bond with cupric ions forming a violetcolored chelate.
Copper
Biuret Method
INTERFERENCE:
lipemic sample
Has the highest analytical sensitivity which
measures small concentration of protein
Folin-Ciocalteu (Lowry) Method
Folin-Ciocalteu (Lowry) Method
Principle: ______ __ _________ ______ such as tyrosine, tryptophan, and histidine.
Oxidation of phenolic compounds
Folin-Ciocalteu (Lowry) Method
tyrosine, tryptophan, and histidine to
give a ?
Deep blue color
Folin-Ciocalteu (Lowry) Method
MOLYBDIC
ACID OR PHENOL REAGENT; oxidize phenolic
compounds
PHOSPHOTUNGSTIC
Folin-Ciocalteu (Lowry) Method
the reagent will reduce which is
measured
spectrophotometrically
Folin-Ciocalteu (Lowry) Method
Color enhancer:
BIRURET REAGENT
The absorbance of proteins at 210nm is due to the
absorbance of the peptide bonds at specific
wavelength.
UV Absorption Method
UV Absorption Method
Majority of proteins has absorbance of 210
except if there is ________ which is absorption at 280nm.
aromatic amino acid
UV Absorption Method
tryptophan, tyrosine, and
phenylalanine absorption at ?
280nm
based on measurement of
refractive index of serum total protein.
Refractometry
Refractometry
Based how protein bend light and the
changes is ______ due to the
concentration of protein in the sample.
proportional
Refractometry
More changes in light = ?
high protein in
sample
Measurement
depends on formation of a uniform fine precipitate
which scatters incident light in suspension
(NEPHELOMETRY) or block light (TURBIDIMETRY).
Turbidimetry and Nephelometry
used for detection of
proteins as little as 1 ug
Coomasie Brilliant Blue Dye
develops violet color by reacting with
primary amines widely used for detection of peptides
and amino acids after paper chromatography
Ninhydrin
Separation technique
Serum Protein Electrophoresis (SPE)
Migration of charged particles in an
electric field
Serum Protein Electrophoresis (SPE)
Serum Protein Electrophoresis (SPE)
cathode
Negative terminus electrode
Serum Protein Electrophoresis (SPE)
anode
Positive terminus electrode
Serum Protein Electrophoresis (SPE)
Protein will migrate in the anode because its ?
amphoteric
Serum Protein Electrophoresis (SPE)
proteins charge is negative
Increase p H(basic)
Serum Protein Electrophoresis (SPE)
the protein charge is positive
Decrease p H(acidic)
Isoelectric Property of Proteins
either positive or negative
depending on pH condition
AMPHOTERIC
Isoelectric Property of Proteins
ph where the
charge of protein is 0
No charge at ISOELECTRIC POINT
The acidic and basic amino acids content of proteins
determines its net charge.
Isoelectric Property of Proteins
Isoelectric Property of Proteins
amine and
carboxylic acid
Positive and negative component
Isoelectric Property of Proteins
pH 8.6 which is basic
BARBITAL (VERONAL)
