Bradford assay how does it work




















There are two types of Bradford protein assay kits. The other kit provides more flexibility with 5x concentrated reagent and lyophilized BSA or bovine-globulin standards, allowing the user to prepare reagent and standards at any concentration. All protein assays are subject to interference by certain substances under some conditions.

The Bradford protein assay is quite robust and is compatible with many compounds commonly found in protein preparations. A standard Bradford protein assay kit is compatible with the following chemicals:. For membrane preparations that have been solubilized with detergents, depending on the type of detergent and the concentration, it may be necessary to dilute the sample to reduce the concentration.

If a quick Bradford protein assay kit with a ready-to-use Bradford reagent is used, detergents that interfere will need to be at a lower concentration in the sample than in the standard assay due to the high sample-to-dye ratio. This assay is based on a single Coomassie dye based reagent.

The binding of protein to the dye results in a change of color from brown to blue. The change in color density is proportional to protein concentration. Protein estimation can be performed using as little as 0. Want more Protein Man blogs? Features Sensitivity: Linear responses over the range of 0. My Account. All Rights Reserved. See how the ready-to-use Quick Start Bradford protein assay can give you fast, easy, and accurate protein concentrations.

This is one of two Coomassie dyes that are often confused. Coomassie R is used to stain protein gels but is not used in protein assays. In addition to being used in the Bradford assay, Coomassie G can also be used to stain protein gels, although it is less sensitive than Coomassie R Under acidic conditions, Coomassie G is cationic, mainly doubly protonated, and is red, whereas in neutral conditions the dye is green, and the anionic form is blue.

The Bradford reagent is an acidified solution of Coomassie G; the dye is thus primarily protonated and red. The basis for the Bradford assay is that in order for the Coomassie dye to bind stably to protein, it needs to be doubly protonated.

When the dye comes in contact with protein, the first electron is donated to charged groups on the protein. This disrupts the structure of the protein, resulting in exposure of hydrophobic pockets. The dye binds to these pockets, with the sulfonic acid groups binding to positive amines.



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