Protein Analysis: Bradford Assay
Basic Principles:
- Spectroscopic Analysis for Protiens
- Spectroscopic Analysis for Protiens
- Coomassie Brilliant Blue Dye G-250
- Amphoteric Measuring
Protocol:
- Create a standard curve using the known 2.0 mg/ml BSA solution and dilute with distilled water
- Make at least 4 separate dilutions (i.e 1 mg/mL, 0.75 mg/mL, 0.5 mg/mL, 0.25 mg/mL).
- Take 100 uL of the dilution and add 3 mL Bradford Dye Reagent and incubate for 5 mins.
- Adjust the spectrophotometer to a wavelength of 595 nm, and blank using just distill water.
- After the incubation, pipette 1 to 1.5 ml of the mixture to each cuvette and record the readings.
- Plot the absorbance of the standards vs. their concentration and find the extinction coefficient to calculate the concentrations of your food samples.
- Now homogenize 1.0 g of your food sample in 10 mL of distilled water and filter with Buchner funnel using a Whatman #1 filter paper.
- Extract 0.1 mL of the filtered homogenized mixture and follow the Bradford Assay protocol mentioned above (3-5).
Results:
- Spinach Dip: 0.597 mg of protein (Nutritional Label: "less than 1 gram")
- Veggie Burger: 1.517 g (Nutrition Label: 5 grams)
- Pickled Herring: 2.522 g
(Nutrition Label: 10 grams)