Bradford method is one of the reliable accurate method to estimation total protein.

Use the following reference which is very useful

Bradford, M.M., (1976); A rapid and sensitive method for the quantification of

microgram quantities of protein utilizing the principle of protein dye binding.

Analytical Biochemistry, 7, 248-254.

Attached doc helpful for you to quantify your total protein.

all the best

Bradford method is one of the reliable accurate method to estimation total protein.

Use the following reference which is very useful

Bradford, M.M., (1976); A rapid and sensitive method for the quantification of

microgram quantities of protein utilizing the principle of protein dye binding.

Analytical Biochemistry, 7, 248-254.

Dear Albert

I recommend Bradford assay because this method is too fast and need no complex apparatus.

Also you can use the spectroscopy method by using the following formula:

Protein concentration (mg/ml)=(Absorbance 280nm *0.54)-(Abs 260nm*0.74)

Lowry method also can be used but this method is not rapid enough and has many steps but detectionlimit is good.

The easiest and quickest total protein assay that does not require any reagents aside from the standard is absorbance measurement at 280 nm. The protocol can be found at

http://www.pharmacopeia.cn/v29240/usp29nf24s0_c1047s198.html.

Bradford is a good compromise ! Be careful with the Lowry method because plant extracts contain phenolic compounds that could interfere with this assay. Alternately you can try to precipitate the proteins with 1 volume of cold absolute ethanol (kept at -20°C), then redissolve, you can avoid background.

All methods have their problems.

*BCA involves the reduction of Cu2+ by peptide bonds. Reducing sugars also reduce copper so can interfere.

*Bradford can cross react with other components in complex plant mixtures, especially detergents, such as phospholipids.

*Absorption at 280 nm measures aromatic amino acids in the protein, but also DNA, RNA and phenolics in the plant material.

*Infra-red absorption is a specific method, detecting the peptide bond shift, but requires specialist equipment, such as the Millipore direct detect.

*Thermal decomposition will measure the specific degradation of sugars, proteins etc, but destroys your sample.

*Quantitative NMR using low field instruments, based on unique peptide shifts, can be accurate but again requires specialist equipment.

It depends on your exact requirements as to which assay is best.

The BCA assay is the one I often use. It is fast and convenient. I prefer to use this instead of Bradford because the BCA assay tolerates the use of several detergents often used to solubilize the cells.

Total Quantification of proteins can be done by several methods:

1. Absorbance at 280 nm

2. Biuret Method or Lowry estimation

3. BCA estimation

4. Bradford method

Each of these have there own advantages and disadvantages.

1. 280 nm is a very quick method but higher concentrations of Nacl interfere in the assay.

2. Oldest and most reliable method but Phenolic compounds interfere in Biuret and Lowry estimation protocols.

3. Quick , tolerant of number of detergents but Reducing sugar, lipids, tyrosine, phenolics interfere in case of BCA.

4. Bradford uses Coomasie Blue which is a dye that binds specifically to proteins. It is very accurate and sensitive, compatible to most buffers, sugars, chaotropic agents but high concentrations of detergent interfere in the assay

So if you wish to quantify protein in the plant sample, you must have an idea that what other components could be present in the sample under test...

Wish you good luck........

Thank you for all your suggestions. I will take a look carefully to all these methods that all you mentioned and I will choose the best one.

Many thanks!

I think that everyone aggrees that the best method is the one that gives accurate and precise results. One should also take into consideration the analytical range of the method especially if a great number of samples should be analyzed. So sometimes it is the nature of sample and the concentration of analyte in it that suggest the best method. If you have plant samples a Lowry method is enough accurate and precise with high dynamic range. If you use some more sensitive methods like Bradford or BCA you will need to make a number of dilutions in order to be inside their analytical range.

You could use bradford-, lawry-, biuret- method or even kjeldahl method but it is for crude protein in sample.

 Here is the best reference to get what you want. It is free and you simply can find it on the Internet.

Bradfod, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Analytical Biochemistry 72, 248-254

2D quant kit method (http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences/80648356)

can remove many interfering substances before color development. Steps include precipitation of proteins using co-precipitating reagents and color development using copper reagent.