Dear,

You can use the Bradford assay, it is one of the easiest spectroscopic methods to obtain an accurate protein quantification.

For info you can read the freely available BioRad manual.

Thank you for your answer. However, Bradford assay quantifies total protein and I intend to quantify just my target protein.

Do you have any standards? In this case you can run a SEC-HPLC and quantify your protein on the basis of a previously built calibration curve.

No, I haven't any and still do not exist any commercial standards.

I may be mistaken but as far as I understand, all of the methods require the protein to first be purified through various rounds of chromatography to ensure purity of the sample. The Bradford assay is fast but can have interference from things like detergents or denaturants and the reaction is also dependent on the amino acid composition which makes it difficult to develop a standard curve. If you have purified your protein you should be able to use the Bicinchoninic acid assay. It's fairly easy and accurate but takes some time. You have to set up a standard curve using a standard of bovine serum albumin (BSA) dilutions then make dilutions of your sample with the BCA reagent and incubate them for about 30 minutes. All of this can be done in a 96 well plate and read in a plate absorbance reader. A calculation is then used to determine the quantity of protein in your purified sample. The idea is that if your sample was purified well enough the readings will be accurate. Again, I may be incorrect but this is my understanding of how to quantify a protein of interest.

If you are interested I can provide you with a BCA protocol that several people have used over the past few years.

Thank you for your answer. However, using BCA method we'll find the same issue: this method quantifies total protein. Even if I use immunoaffinity chromatography or other specific method I can't completely ensure that the purified sample only has my target protein.

You might be interested in this webinar.

https://www.chromacademy.com/Amino-Acid-Analysis-in-Spent-Media.html

Hello,

I would say that for now your major obstacle is the purity of the protein. If you reach to a point where it is pure, you can use both the methods suggested in other answers. I assume you have purified your protein through preparative chromatography..however, have you explored other stationary phases? For example preparative-HPLC (e.g. C18, or C18 with other groups) stationary phases that might enhance your selectivity. The fraction purity can then be confirmed by LC-MS or LC-MS/MS. Only after confirming that the fractions are pure you can quantify the protein (BCA and Bradford), giving you don't have a commercial standard or response factor towards other proteins.

If it is a pure protein solution (only your protein of interest present) then you can run a UV 280 after obtaining the molar absorption coefficient from Protparam. Also you can run the purified protein sample on an SDS page with known concentrations of standard protein on adjacent wells and use a software to quantify.

As far as I know: purify all proteins, run SDS-PAGE if you know size of your protein, and then f.ex. BIO-1D++ software can measure absorbance profiles and compute individual protein portions.

I`m about to work out patatine detection in potato tubers and would appreciate if anyone who has experience could comment and give advice :)