Bradford Protein Assay (By using #5000006)

Bioreagents:

The Bradford assay is one of the colorimetric methods used to measure the total protein concentration in solutions. It is widely used as an indicator for protein concentration. It entails the binding of Coomassie Brilliant Blue G-250 dye with proteins, which pushes the shift of the maximum absorption wavelengths from 465 nm to 595 nm. The Bio-Rad Protein Assay Concentrate (Dye Reagent #5000006) has developed as a respected commercial kit for completing the Bradford assay. It contains a dye reagent into which it is added and also there is bovine serum albumin (BSA) standards.

Key Components

Stain Reagent Concentrate (has Coomassie Brilliant Blue G-250 combined with methanol and phosphoric acid).

Pierce™ Coomassie (Bradford) Protein Assay Kit

Here you have to quantify the hemoglobin content against the Bovine Serum Albumin (BSA) Standard.

Protocol

Use the doing buffer for the stock solution and the same one for the unknown samples to prepare diluted BSA standards from 2 mg/ml stock solution.
The process is done by pipetting 20 μL of each standard to one microplate well and the unknown sample to the other microplate well.
Gently pipette 200 μL of the diluted 1X Dye Reagent to each well. Mix cells very gently.
After removing the frozen product from a freezer, place it on a counter for cultivation at room temperature for 5 mins.
Leave the well plate in the plate reader for about 4 minutes and measure the absorbance at 595 nm.
Empty the value 595nm (11.31 average) from all additional measurements.
Slope-intercept plot of mean blank-corrected 595 nm absorbance measured for each BSA standard versus its concentration in μg/mL should be drawn.
Subsequently, measure the standard curve to determine the protein concentration of the unknown samples.

The Bradford assay is fast, easy and inexpensive to do, hence it is appropriate for those who require high-throughput of the gels. Nevertheless, the UV-Vis assay is often more prone to interference from some compounds compared with the BCA assay, and could also demonstrate variation in color response between proteins.

Preprint

Preprint

Bradford Protein Assay (By using #5000006)