Practical Session 4 Flashcards
What is the title and aims of practical 4?
Colorimetric Assay of Serum Protein
- Make a standard curve for protein serum and calculate ε for this compound.
- Use micropipettes.
- Understand the prediction error from results calculated from callibration curves.
How is the biuret test known?
Piotrowski’s test.
What is the biuret test?
A chemical test used for detecting peptide bonds.
What does a copper (ll) ion form when peptides are present?
Mauve-coloured coordination complexes in alkaline solution.
At which wavelength does the blue adduct absorb?
540 nm = λ.
What do biuret and peptide bonds form when they react with cupric ion?
A blue adduct.
What does the biuret reagent include?
Sodium potassium tartrate.
What does sodium potassium tartrate do?
Stabilize cupric ions that are sensitive to reduce to Cu+.
How is the biuret reaction characterised?
The most common technique for measuring total protein using automated chemistry analysers.
What do modifications of the biuret method include?
Lowry and Folin-Ciocalteu techniques.
Where are the Folin-Ciocalteu techniques useful?
Detecting minute amounts of protein presented after isolation/separation.
What does the measurement of total protein determine?
Concentration of all proteins present in serum (except clotting factors).
Which are the major proteins?
Albumin.
Immunoglobulins: IgG, IgA, IgM.
How is the total protein measurement used clinically?
- Total protein measured in serum –> gives indication of total immunoglobulin concentration: total protein - albumin = globulins.
- Total protein included in ‘liver function tests’: chronic liver diseases –> increase immunoglobulin –> increase total protein.
- Total protein measured in suspected humoral immunodeficiency: diagnosis by immunoglobulins measurements.
- Total protein measured when a patient is suspected of having paraprotein but not high: serum protein electrophoresis.
What is total protein measurement without measurement of albumin?
Of little value.
What is the proportion of light absorbed by a solution of a particular compound?
A function of the concentration of the compound.
What does spectrophotometry allow?
Quantitative analysis of concentration of a substance from Beer-Lambert relationship.
What does a spectrophotometer do?
Direct light of a specific wavelength on solution.
Calculate absorbance.
What is the light from the spectrophotometer?
The incident light.
What is the light that passes through the solution?
The transmitted light.
What is the absorbance of the solution?
The log of the ration of the incident light divided by transmitted light.
A (Absorbance) = log10 (Intensity of incident light (Io) / Intensity of transmitted light (I))
How can we find the concentration of the solution?
Know absorbance.
Beer-Lambert equation.
What is the Beer-Lambert equation?
A = ε.l.c
What does the Beer-Lambert equation mean?
A = ε.l.c
ε = Absorbance of 1M solution of substance measured through 1cm light path, constant.
c = concentration, moles/litres.
l = length of light path through solution, cm = 1 cm.
