What is the composition of the Bradford reagent?

What is the composition of the Bradford reagent?

The Bradford reagent is an acidified solution of Coomassie G-250; 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.

How do you make 1x Bradford reagent?

Bradford’s Reagent Recipe

1. Dissolve 50mg of Coomassie Blue G250 in 50ml of methanol.
2. Add 100ml of 85% H3PO4 to the solution from step 1.
3. Add the solution from step 2 into 500ml of H2O and mix.
4. Filter to remove and precipitates.
5. Add an additional 350ml of H2O.
6. Store at 4oC.

What does Bradford reagent bind to?

protein
Chemistry of Bradford, Coomassie-based protein assays In an acidic environment, proteins bind to coomassie dye. This results in a spectral shift from the reddish brown form of the dye (absorbance maximum at 465 nm) to the blue form (absorbance maximum at 610 nm).

How do you do the Bradford method?

Procedure

1. Bradford reagent: Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol, add 100 ml 85% (w/v) phosphoric acid.
2. (Optional) 1 M NaOH (to be used if samples are not readily soluble in the color reagent).

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 do you calculate Bradford assay concentration?

To calculate the concentration of the undiluted, unknown sample, simply multiply by the dilution factor. So, 0.5 x 10= 5mg/ml.

How do you store Bradford reagents?

The product is stored at 2–8 °C. It is stable at 2–8 °C in an unopened container for at least 1 year. The standard 3.1 ml Bradford assay consists of mixing 1 part of the protein sample with 30 parts of the Bradford Reagent.

How do you analyze Bradford assay results?

Perform the assay and calculate the standard (see below). The result should be around 0.5mg/ml. To calculate the concentration of the undiluted, unknown sample, simply multiply by the dilution factor. So, 0.5 x 10= 5mg/ml.