How to Prepare Bradford Reagent
The Bradford protein assay is a widely used method for determining protein concentrations in a solution. It is known for its simplicity, sensitivity, and lack of interference from common ions. The assay is based on the binding of Coomassie Brilliant Blue G-250 dye to proteins, resulting in a color change that can be measured spectrophotometrically. In this article, we will guide you through the process of preparing Bradford reagent, a crucial component of this assay.
Materials Needed
Before you begin, gather the following materials:
– 100 mL of 95% ethanol
– 10 g of Coomassie Brilliant Blue G-250
– 2.5 mL of glacial acetic acid
– 10 mL of 85% phosphoric acid
– Distilled water
– A 1000 mL beaker
– A glass rod
– A funnel
– A filter paper
– A storage bottle
Preparation Steps
1.
Mix the Ethanol:
In a 1000 mL beaker, pour 95% ethanol and stir well with a glass rod to ensure complete mixing.
2.
Add the Dye:
Carefully add 10 g of Coomassie Brilliant Blue G-250 to the ethanol solution. Stir the mixture thoroughly until the dye is completely dissolved.
3.
Add Acetic Acid:
Slowly add 2.5 mL of glacial acetic acid to the dye solution while stirring continuously. Be cautious, as glacial acetic acid is highly corrosive.
4.
Add Phosphoric Acid:
Add 10 mL of 85% phosphoric acid to the solution, again while stirring continuously.
5.
Filter the Solution:
Use a funnel and filter paper to filter the mixture to remove any undissolved particles.
6.
Transfer to Storage Bottle:
Pour the filtered solution into a storage bottle and seal it tightly. Store the prepared Bradford reagent in a cool, dark place to maintain its stability.
Storage and Usage
It is essential to store the Bradford reagent properly to ensure its effectiveness. Keep it in a cool, dark place, away from direct sunlight and heat sources. The reagent has a shelf life of approximately six months when stored correctly.
When using the Bradford reagent, prepare a standard curve using known protein concentrations. Then, add the protein sample to the reagent and measure the absorbance at 595 nm. The protein concentration can be determined by comparing the absorbance of the sample to the standard curve.
In conclusion, preparing Bradford reagent is a straightforward process that requires attention to detail and proper handling of chemicals. By following these steps, you can ensure the accuracy and reliability of your protein concentration measurements using the Bradford protein assay.
