Bradford Protein Asssay

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  • Topic: Bicinchoninic acid assay, Bradford protein assay, Amino acid
  • Pages : 2 (393 words )
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  • Published : November 6, 2010
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Question 5:
The chromophore in this assay is Coomassie Brilliant Blue dye. Question 6:
It is important to set up a blank to separate the solute (saline) from the protein (stock). By subtracting the absorbance of the blank (which has no protein present) from the original absorbance the absorbance of the protein at each concentration will remain.

Question 7:
The Lowry method relies on two different reactions. The first is the formation of a copper ion complex with amide bonds, forming reduced copper in alkaline solutions. This is called a "Biuret" chromophore. The second is the reduction of Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate) by tyrosine and tryptophan residues. The reduced Folin-Ciocalteu reagent is blue and therefore is detectable with a spectrophotometer in the range of 750 nm. Using the Folin-Ciocalteu reagent to detect reduced copper makes the assay nearly 100 times more sensitive than the Biuret reaction alone. The BCA Assay:

The BCA assay is a two step assay, in the first step Cu2+ is reduced to Cu1+ by forming a complex with protein amide bonds. In the second step, Bicinchoninic acid (BCA) forms a purple complex with Cu1+ which is detectable at 562nm. this assay is relatively slow unless heated. When comparing these methods to the Bradford assay, the Lowry method has many interfering substances, it is a slow reaction and the proteins can irreversibly denature. The BCA assay, is also a slow reaction and requires more time ranging from 40minutes to 24hours.

Question 8:
UV absorption can determine protein concentration, most Proteins, absorb ultraviolet light relatively strongly. It is the amino acids in the proteins that absorb the UV light. The strong absorbance of UV light by protein allows for rapid analysis of protein samples. The optical absorbance of protein is measured at 280 nm. At this wavelength, the absorbance of protein is largely due to the amino acids tryptophan, tyrosine and cysteine .The...
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