Question

In: Chemistry

Compare and contrast the BCA and Bradford methods for protein concentration determination. How does each work?...

Compare and contrast the BCA and Bradford methods for protein concentration determination. How does each work? Give an example of something that interferes with these methods and explain where each works better than the other. Be as specific as possible.

Solutions

Expert Solution

BCA method
Bradford method
Based on Protein-copper chelation
Based on Dye-binding based detection
Here, the peptide bonds in protein reduce Cu2+ ions from the copper(II) sulfate to Cu+. The amount of Cu2+ reduced is proportional to the amount of protein present in the solution. Next, two molecules of bicinchoninic acid chelate with each Cu+ ion, forming a purple colored complex.
A colorimetric protein assay, based on an absorbance shift of the dye Coomassie Brilliant Blue G-250. The ability of Coomassie blue to bind protein causing the dye to shift from a red color to a blue color.
Assay is more susceptible to interference by various chemicals that may be present in protein samples
Bradford protein assay is less susceptible to interference by various chemicals that may be present in protein samples
Interfere in the presence of cysteine/cystine, tyrosine, and tryptophan side chains, carbohydrates, catecholamines, lipids, phenol red, impure sucrose or glycerol, uric acid, iron and hydrogen peroxide, etc.
Interfere at the presence of Sodium dodecyl sulfate (SDS)
Absorbance at 562 nm
Absorbance at 595 nm
Can determine protein quantities between 0.5 μg/mL to 1.5 mg/mL
Can determine protein quantities as little as 1 to 20 μg
Sensitive
Extreamely sensitive. Bradford reagent stains the plastic.
More time consuming
Less time consuming

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