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Protein Quantification Kits on ZAGENO

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.

Expand your understanding in our Knowledge Section. Perfect your experiments with our Troubleshooting Guides. Join in the discussion within our Community. Learn about ZAGENO at How It Works.

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.

Lowry Assay

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.

Compare Bradford Assay Products!

With our compare function, you can avoid all the time and energy wasted sifting through multiple web pages from different suppliers. At ZAGENO you can clearly see kits side-by-side, with the relevant attributes for each kit neatly in line for easy selection of the best product for you.

Click on the comparison below for a clearer view!

This example highlights how every kit has positive and negative aspects, and that only side-by-side comparison can allow you to correctly judge which is the right kit for you.

For instance, this comparison shows:

  • that the Bradford Protein Assay Kit (OZ) is the cheapest per preparation
  • that the Bio-Rad Protein Assay Kit I can use the largest amount of sample
  • that the CB Protein Assay requires no more than five minute incubation time, whereas the other two may require longer

The ZAGENO comparison engine does not show the best kit, but instead helps you, the researcher, choose the most sutiable kit for your experiment.

Go to our How it Works page for a guide to using the ZAGENO comparison engine.

Video Credits

Video: teachinhawaii/YouTube


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