Analytical Methods Flashcards
What are the different methods we can use to measure Proteins?
Kjeldahl
Dye Binding
Biuret
Direct Photometric
Turbidimetric/Nephelometric
Electrophoresis
Kennels Do Burn Dont Touch Neever
What is the classical method of Protein testing?
Kjeldahl Method
How is the Kjeldahl test carried out?
Tests for nitrogen present
Nitrogen converted to Ammonium using Acid Digestion
Quantified using Titration
Multiplied by 6.25
What is important about the Kjeldahl Method?
It is highly accurate and reproducible but is it too time consuming
It now validates reference materials
What relies on the shift in Absorbance Spectra of dyes when binding occurs?
Dye Binding Method
What is important about the Dye Binding Method?
Variable Binding Affinity
Limited interference
What are the common dyes used in the Dye Binding method?
Bromocresol Green/Purple
Coomassie Brillian Blue
Ponceau. S
Lissamine Green
What is based off measuring the reaction between Peptide bonds and Cu2+ ions in alkaline conditions?
Biuret Method
How is the Biuret Method carried out?
Cu2+ binds to peptide bonds causing a change in color
Color change is proportional to Peptide bonds present
What is important about the Biuret Method?
Reagent is a blue color due to Cu2+
Interference from Bilirubin, Hemolysis, and Lipemia
What measures protein content by absorbance of UV light?
Direct Photometric
What is important about Direct Photometric?
Uric Acid and Bilirubin absorb UV light ~280nm
May require removal of low weight items using Gel Filtration
What is widely used to asses High Abundance Proteins and forms Aggregates?
Turbidimetric and Nephelometric
What is the difference between Turbidimetric and Nephelometric?
Turbidimetric measures absorbance caused by Aggregates
Nephelometric does the same but reads reflected light at an angle
What can be used to measure protein content in Serum, Plasma, Urine, and CSF
Electrophoretic Techniques
