Quantifying Protein in a Sample
Quantification of protein concentration is a necessary first step during any protein analysis. There are various detection methods to achieve this, ranging from simple UV absorbance measurements (as amino acids exhibit strong UV-light absorption), to HPLC. However, the more cost effective technique is through the use of simple colorimetric assays.
The Bradford method
One technique to determine protein concentration and the amount of sample is the Bradford method. Its principle: negatively charged Coomassie® Blue dye binds to positively charged proteins. When no proteins are present in the sample, the dye absorbs at 465 nm and has a red color. However, when proteins are introduced, the color changes to blue and absorbs at 595 nm. This absorption can then be cross-referenced to a standard curve. However, Bradford assays are often inhibited by detergents and can be difficult to read at certain concentrations. Thus, the method for protein quantification used will have to be curtailed to your experimental needs.
Our Bradford Assay Troubleshoot will help solve any issues you encounter.
BCA Protein Assay
The BCA Protein Assay uses bicinchoninic acid (BCA) to colorimetrically detect the color change produced by the cuprous cation (Cu1+), which results from the reduction of Cu2+ to Cu1+ by protein in an alkaline medium.
In the biuret reaction, copper chelates with protein in an alkaline solution to form a light blue complex; peptides with three or more amino acid residues form a chelate complex with cupric ions in the presence of sodium potassium tartrate.
Two molecules of BCA then chelate with one reduced cuprous cation to form an intense purple-colored product. The BCA/copper complex is water-soluble; producing a strong absorbance at 562 nm, proportional to protein concentration. The BCA reagent is 100 times more sensitive than the original pale blue color.
The reaction that leads to BCA color formation is strongly influenced by four amino acid residues (cysteine, tyrosine, and tryptophan). However, the peptide backbone also contributes to color formation, minimizing variability caused by differences in protein composition.
The Lowry protein assay uses cupric sulfate and tartrate in an alkaline solution, which forms tetradentate copper protein complexes when protein is added. Folin-Ciocalteu Reagent is reduced proportionally to these chelated copper complexes, producing a blue water-soluble product which can be measured at 750nm.
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