Bradford's reagent reacts primarily with arginine residues and secondarily with a few other amino acids. The Bradford reagent is an acidified solution of Coomassie G-250; the dye is thus primarily protonated and red. Ed Harlow and David Lane; This protocol was adapted from “Protein Techniques,” Appendix II, in Using Antibodies.

Composition: Bradford reagent consists of Coomassie Brilliant Blue G-250 in phosphoric acid and ethanol. Recipe. Bradford Assay (Bradford Reagent) ... Proteins are diverse in their composition and structure, and with some assays the proteins’ differences in amino acid sequence, isoelectric point (pI), secondary structure, and side chains or prosthetic groups result in variation in the colorimetric response. Features and Benefits • The reagent is ready to use. This assay is used for protein quantitation.

Finally, Bradford reagents result in about twice as much protein-to-protein variation as copper chelation-based assay reagents. NOTE: Coomassie must be dissolved in the methanol FIRST before the other ingredients are added.

Bradford Reagent (5X concentrate) 100 mg Coomassie Brilliant Blue G-250.

Caution: Always add acid slowly into water and do not add water into acid. It is relatively accurate for most proteins, except for small basic polypeptides such as ribonuclease or lysozyme. INTRODUCTION. The coomassie blue dye associates with basic and aromatic amino acids, thereby causing shift in absorbance during protein determination. 47 ml Methanol (100%) 100 ml Phosphoric Acid (85%) QS to 200 ml with H2O. Bradford Reagent Catalog Number B6916 Storage Temperature 2–8 °C TECHNICAL BULLETIN Product Description The Bradford Reagent can be used to determine the concentration of proteins in solution.

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. Coomassie Plus (Bradford) Protein Assay Detergent Compatible Bradford Assay Coomassie (Bradford) Protein Assay; Features: Ready-to-use, reducing agent–compatible Bradford assay reagent that provides increased linearity of response and only half the protein-to-protein variation of other commercial Bradford assay formulations This makes it good for the quick estimation of protein concentration in a crude mixture, but not very good for estimation of a purified protein that is acidic or basic.

The reagent does not keep well and it is therefore advisable to make it up when it is actually required. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1999. Bradford Assay.

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