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In my various laboratory experiences (N=5; probably not enough to achieve any kind of statistical significance) it seems that labs either use a Bradford or a BCA protein assay to quantify protein concentrations.

Both assays come in convenient kits that can be performed in micro, multiwell plates or in a standard, larger volume format.

So how does a lab choose?

micro, multiwell plate, and standard assays

Bradford protein assay

The Bradford assay is based on the ability of Coomassie blue (you know, they dye you use to stain your protein gels) to bind protein causing the dye to shift from a red color to a blue color. This shift can be quantified by measuring the absorbance of your samples at 595 nm.

The Bradford assay is quick – samples can be read 5 minutes after the addition of the dye to your sample.

A lot of people like the Bradford assay because fewer substances interfere with the assay (including reducing agents) – with the notable exception of high concentrations of some detergents. Which can be a problem if you require a lot of detergent to lyse your cells.

In addition, sometimes your samples need to be diluted before measuring them with Bradford leading to final calculation confusion.

Warning – don’t plan on washing and reusing your cuvettes. The Bradford reagent stains the plastic.

BCA (bicinchoninic acid) protein assay

The BCA assay is based on the traditional Lowry assay except bicinchoninic acid is used resulting in a very pretty purple color that absorbs at 560 nm.

BCA is sensitive and has a broad dynamic range – capable of measuring protein concentrations of 0.5 μg/mL to 1.5 mg/mL

A depressing list of things can interfere with BCA, including, carbohydrates, catecholamines, tryptophan, lipids, phenol red, cysteine, tyrosine, impure sucrose or glycerol, uric acid, iron and hydrogen peroxide – but truthfully I have never found this to be a problem with general samples in the lab.

BCA is not compatible with reducing agents and you also have to invest some time in the assay as samples are read after 30-60 minutes of incubation.

Based on this information though, I wonder – why should a lab choose?

Why not use the Bradford for a quick assay to measure less concentrated proteins, or those in buffers containing reducing agents and turn to the BCA for highly concentrated samples lysed in detergent?

We are curious – do you prefer one assay over the other?

Take the poll to tell us what you use and leave us a comment to tell us why.

 

Photo courtesy of West Point – The U.S. Military Academy

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4 Responses to “Which Protein Assay Camp Are You In? Bradford vs BCA”

  1. Ben Roman

    Hello,

    I recently used the Bradford assay to measure protein in dried duckweed powder. The results for protein content from the Bradford assay is low compared to a combustion method in the same biomass (35% from combustion, 20% from Bradford assay). A colleague of mine recommended that I use the BCA assay, because he had more success with this assay in the past. I’m curious if you’ve found anything to support my colleague’s claim. If you could let me know what the general consensus from the poll is, I would greatly appreciate it. Thank you!

    Best,

    Ben Roman
    Ph.D. Candidate, Environmental Engineering
    Department of Civil and Environmental Engieering
    The Pennsylvania State University

    Reply
    • Sia Ghazvini

      Dear Ben,

      Both the Bradford and the BCA methods are good choices if you want to accurately measure the protein content of your sample. You will need to select the assay based on what else is in your sample. Each method has a list of compatible substances that you should check before proceeding. Since you will likely use a standard that is prepared from BSA you will have some issues if the dye binds your protein sample differently.

      Regards,
      Kim

      Reply

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