Proteins part 4 Flashcards
What is the product color of Biuret method?
Purple color
- Copper binds to the peptide bond
Biuret method
- Protein + CuSO4 + NaOH + Rochelle salt = ____________
PURPLE CHROMOGEN
Biuret method is based on the formation of the violet-colored chelate between cupric ions and requires at least ______ peptide bonds. Measured at __________
2
540nm
In this method, molecules are formed when Urea is heated at 180◦C.
Biuret
- 2 molecules of urea form _____ molecule of biuret
one (2:1)
__________ is preferred for Biuret reaction
Kinetic Biuret method
These will not be detected using a Biuret method.
Amino acid and Dipeptide
What are the reagents used in Biuret method?
a. Alkaline Copper Sulfate
b. Sodium Hydroxide
c. Rochelle’s salt
d. Potassium iodide
c. Rochelle’s salt = ___________________
Sodium Potassium Tartrate
- Used to complex Cupric ion and prevent their precipitation
Rochelle’s salt
- Keeps Copper in the Cupric form
Potassium iodide
- These 3 causes false increase in the range of Biuret method
a. High bilirubin
b. Elevated lipids
c. Hemolyzed samples
- Causes false decrease levels
d. High Blood Ammonia levels
- Tyrosine & Tryptophan reduce PT-PMA reagent (folin-ciocalteu) to give a ______________
BLUE COLOR
These reduces PT-PMA reagent
Tyrosine and Tryptophan
- F-CTM or Lowry Method detects proteins in low concentration, how low?
10-60ug/mL
- Detects proteins in low concentration (10-60 ug/mL)
FOLIN-CIOCAL TEU METHOD / LOWRY METHOD
- Widely used in research to measure tissue and enzyme proteins
FOLIN-CIOCAL TEU METHOD / LOWRY METHOD
- Not satisfactory for urine and CSF protein determination
FOLIN-CIOCAL TEU METHOD / LOWRY METHOD
In Folin-ciocal Teu method, the specimens are mixed with an _______________ followed by the addition of the_____________
Alkaline Copper solution
Lowry method.
composed of phosphotungstic acid + molybdic acid which are used to reduce tungsten blue by copper complex with peptide and by tyrosine and tryptophan
Phosphotungstomolybdic acid
Phosphotungstomolybdic acid is composed of?
phosphotungstic acid + molybdic acid
What do phosphotungstic acid + molybdic acid reduce?
tungsten blue by copper complex with peptide and by tyrosine and tryptophan
In Lowry method, The absorbance product are measured at a wavelength of ________. Dependent on the phenolic groups.
650-700nm.
Due to the high electron density of aromatic rings of tyrosine and tryptophan in solution (pH 8)
ABSORPTION OF UV LIGHT AT 280nm
Employed to stain protein bonds after electrophoresis
Dye-binding methods
Analysis of fluid with lower protein concentration such as urine and CSF
Pyrogallol red
Is the most sensitive, specific and the most precise among the dye-binding assays
Bromcreasol purple
Albumin at pH 4.2 is cationic and binds with anionic dye
BCG
It is the most commonly used dye and has to measure the absorbance
BCG
- Is used in urine reagent strips
- Sensitive to Albumin
Tetrabromphenol blue
Sensitive to AA
Ninhydrin
HABA refers to?
(2,4-dihydroxyazobenzenebenzoic acid)
Rapidly estimates protein at high concentrations. Often used in urine specimen.
REFRACTOMETRY
Turbidimetry and Nephelometry is AKA as?
Salt precipitation
What are the precipitating agents of Turbidimetry and Nephelometry?
SSA
Trichloroacetic acid
Precipitates all forms of proteins
SSA
Precipitates Globulins than Albumins
Trichloroacetic acid
This method are often used for measuring urine and CSF protein
Turbidimetric
Can used _______ Chloride under the alkaline condition and ________________ under alkaline conditions
Benzethonium
Benzalkonium salts
Determines the movement of proteins during the alkaline electrophoresis from the origin towards the ANODE.
PROTEIN ELECTROPHORESIS
protein donates protons = (-) charge. Therefore migrates towards Anode
Alkaline ph
Protein receives protons = (+) charge
* Migrates towards the cathode
Acidic pH
