2. Protein Purification: Visualization Flashcards
How does UV spectrophotometry work?
It depends on phenylalanine, tryptophan, and tyrosine which are aromatic groups that absorb UV light.
What are the advantages and disadvantages of UV spectrophotometry?
Advantages: it is simple and fast and samples can be recovered.
Disadvantages: interference from other chromophores; specific absorbance values for specific proteins being handled have to be known; if there is a nucleic acid, contamination, absorbance readings will be significantly higher at 280nm.
How does BCA work?
The protein reacts with cupric ion which then gets reduced into cuprous ion, under alkaline conditions. BCA chelates with cuprous ion to form multiple bonds that form ring like structures which absorb light at 562nm to give a purple colored complex.
How does Bradford assay work?
Proteins with basic or aromatic side chains bind with Coomassie blue dye via hydrophobic or Van der Waal’s interactions to form protein-dye complex which turns from reddish brown at 465nm to blue at 595nm.
How do substances interfere with colorimetric assays?
Different assays have different substances with different thresholds that may react with different substances in the sample. Once threshold of substance is reached, inaccurate readings may occur.
Compare BCA and Bradford in terms of variability, time, substance compatibility, and detection range.
Variability: BCA less than Bradford.
Time: BCA slower and more steps.
Substance compatibility: BCA with detergents; Bradford with reductants.
Detection range: BCA 20-2000ug/ml (better range); Bradford 1-1400ug/ml.
