What are the reagents for Bradford assay?
The Bradford reagent is an acidified solution of Coomassie G-250; the dye is thus primarily protonated and red. The basis for the assay is that in order for the Coomassie dye to bind stably to protein, it needs to be doubly protonated.
How do you make Bradford solution?
Bradford reagent: Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol, add 100 ml 85% (w/v) phosphoric acid. Dilute to 1 liter when the dye has completely dissolved, and filter through Whatman #1 paper just before use.
What dye is used in the Bradford assay?
Bradford assay principles Use of Coomassie G-250 Dye in a colorimetric reagent for the detection and quantitation of total protein was first described by Dr. Marion Bradford in 1976.
How do you make a 5x Bradford reagent?
2.2.1. To perform the macro assay dilute the 5x Bradford Reagent 1:5 (1 part by volume plus 4 parts by volume H2O bidest.) and filter the solution. The 1x Bradford solution could be stored for 1 week at RT. To create a calibration curve the reference protein should be diluted as follows: 100, 125, 250, 500, 1000 µg/ml.
How much Bradford reagent should I add?
Add 40 microliters of Bradford reagent to the second well of each pair (the 1/100 dilution), and pipet in and out 8-10 times to mix well.
What color is the Bradford reagent?
reddish brown
The Bradford reagent has Color is reddish brown with lambda max 470 nm. With proteins it develops blue color hence used for colorimetric estimation of proteins using Beer-Lambert law i.e. absorption is proportional to the concentration of the solution.
How do you dilute Bradford reagent?
The Bradford reagent (stain in phosphoric acid and a water/methanol mixture) is designed to be diluted 5-fold with protein. Therefore, in each well we’ll put 160 microliters of protein, diluted in water; to this, we’ll add 40 microliters of Bradford reagent, thereby achieving the recommended 5-fold dilution of reagent.
Is Bradford reagent hazardous?
Hazard statement(s) H290 May be corrosive to metals. H314 Causes severe skin burns and eye damage. H370 Causes damage to organs. Precautionary statement(s) P234 Keep only in original container.
Is Bradford reagent light sensitive?
Bradford is also sensitive to various common chemicals in the solution, including detergents and caotropic agents. UV is sensitive to anything that absorbs UV light (nucleic acids, nucleotides, aromatic substances…)
Why do you dilute Bradford assay?
The protein standard or unknown must be sufficiently diluted not to interfere with the low pH achieved by the acid in the reagent.
What is the quick start Bradford assay kit?
The Quick Start Bradford assay kit is a simple one step procedure. with no need to dilute standards for determining the concentration of protein in a solution. The Quick Start™ Bradford protein assay is a simple and accurate procedure for determining the concentration of protein in solution.
How do I determine protein concentration using the quick start Bradford assay?
Use the Quick Start Bradford protein 1x dye reagent for one-step determination of protein concentration. Many detergents and basic protein buffers interfere with the Bradford assay; interference may be caused by chemical-protein or chemical-dye interactions.
What can interfere with the Bradford assay?
Many detergents and basic protein buffers interfere with the Bradford assay; interference may be caused by chemical-protein or chemical-dye interactions. Refer to the list of compatible reagents for more information.