Bradford is fast but sensitive to the presence of some interfering agents. The most accurate one is Kjeldahl assay which measures total nitrogen.

I agree with the two previous answers. Bradford is a better option as it is much more sensitive than other methods. Qubit® 2.0 Fluorometer is also sensitive and much more convenient. If you go for Qubit, you would just require 1 microlitre of your protein solution for analysis.

The most precise method to quantify protein is amino acid analysis, but it is quite expensive

Many thanks for all the answers. I agree with Reynaldo that amino acid analysis should be the best method. However, the main disadvantage with this method is that at least with the amino acid analysis method we use (the Waters AccQ-Tag derivatization kit with UPLC), you cannot quantify tryptophan and cysteine. Hence, you cannot sum up all the 20 amino acids to get your total protein amount.

Has anyone acquired expected results with amino acid analysis and deriving concentrations for tryptophan and cysteine based on their fraction in the proteome sequence to other amino acid's fraction? Or are there any other "tricks"?

Hi Kaspar and all,

According to my own experience, using Dumas method (total combustion of the sample at 900°C and quantification of N2 in gazeous form), gave the most accurate results for microbial and yeast protein content estimation. The value you get will be multiplied by a factor (6.25 - 6.38), which will give the protein content.

The problem with TKN (Kjeldahl) is that it will consider all the ammoniacal N, as per Armando.

The problem with Bradford and Biuret methods is that you need to release all the protein from their matrix (cell membrane) by a lysis protocol (you may face an incomplete lysis and consequently underestimate your protein content).

Using Leco combustion method requires the Leco device... and also, one must wash the cells before running the analysis.

In summary, dosing protein in microbial biomass is not an easy issue. All the methods proposed here are discussable.

Hope it will help

Amine

Thanks for the answer. We have never used the Dumas method, sounds interesting. One question, though - how will I determine which value use for the factor - you wrote a range there?

Hi Amine,

I dig into the details of the Dumas method and it seems to me that it has the same limitation as Kjeldahl since it quantifies the total nitrogen, not only protein-bound. Is that correct? If yes, then how do you convert the total nitrogen signal into total protein concentration since the distribution of cellular nitrogen varies between different environmental conditions? Thanks;)

Hi Kaspar,

Yes indeed, it does quantify total N but not peptidic bounds.

Therefore, a recognized and standardized conversion calculation allows one to estimate protein content from total N dosage, which is multiplying your N-tot value by 6.25 (personnaly I use this factor value).

Unfortunately, as it is quite complex to estimate protein content in a biomass, using destructive methods such as Dumas or Kjeldahl is a good way to assess the dosage.

If you take a look at publications where protein content is estimated in a microbial biomass, you'll see that they are using these methods.

Hope it will help!

Amine

Hi Amine,

Thanks for the answer. Still, it seems to me that the weakness of these methods is the necessity to use a conversion factor. This is exactly what you do not want to have in a quantitative assay.

Like I said, the distribution of cellular nitrogen varies between environmental conditions where the cells grow. So, I should exactly know which value to use. How have these values been determined and based on which criteria you should choose the value?

Thanks!

Kaspar,

You're right. However, this way of biomass protein quantification is widely recognized by the scientific community. It was determined empirically and are recognized by organisms such as AOAC for example.

I found an interesting document explaining how the so called conversion factor (or Nitrogen Factor) is determined.

As you would see in this document, advantages and limitations are briefly discussed.

Good luck

here is the link: http://www.nrel.gov/docs/gen/fy08/42625.pdf

Hi Amine,

Thank you very much for the reference. However, I have to say that we are back to step one here since as far as I understood the document, calculation of the conversion factor is based on amino acid analysis which pitfalls we discussed above. Hence, it seems to me that performing Kjeldahl or Dumas will not make things better since I would still rely on amino acid analysis.

Any other ideas, anyone?

Yes, they are.

cheers!