Bradford Assay

[perivar]

Hello. i have some question regarding use of bradford method.

 

why is linerization of the bradford method more uncertain near 0 mg/mL concentration of protein and above 0,9 mg/mL where it bends off. In an experiment, the regressive line usually never goes by (0,0) which i assume has something to do with the uncertanty.

 

Using the UV - method give a more "perfect" regressive line with high precision, but if measuring contaminated protein, often gives an overestimation of the concentration, where the bradford method gives a more correct assumtion of the concentration but with lesser precision. Why is the precision lesser with bradford method?

[CharonY]

It is a matter of dynamic (or linear) range which is actually highly dependent on the protein you look at (i.e. binding of the dye or UV absorption vary with the amino acid composition). For virtually all analytical procedures you get a more or less sigmoid curve. At the lower end the non-linear part is partially caused by the lower detection limit. Likewise, at the higher end signal saturation can become an issue.

The part between those which is linear (or exponential) can really be used for quantitative analyses.

This is true for Bradford as well as UV. You may not see it in UV as the dynamic range is often larger so if your measurements are far from the detection limit it will appear to go through zero.

But if you sample close to the detection limit, you will see something similar to Bradford.

 

Regarding precision, that is highly dependent on the type of sample. For crude samples Bradford is more robust and (if timed correctly) has therefore higher precision. For purified low-complexity samples UV is generally easier to handle and has mostly precision advantages due to reduced user-errors.