Berglund:Bradford Assay

BRADFORD ASSAY for protein concentration
(notebook #3 p.4.33)

Materials

Bradford Reagent:
100mg Coomassie Brilliant Blue G-250 (NOT R, nor a different number)(fc = 0.01%)
50mL 95% Ethanol (fc = 4.75%)
100mL 85% phosphoric acid (fc = 8.5%)
• Add water to 1L
• Filter through Whatman #2 filter paper
• Store at 4˚C
• Use within 1 month
• (Can scale down this recipe - I make 50mL reagent at once)

[Or, use Bio-Rad reagent with the below method. It contains methanol, so don't dump that one down the sink.]

Standards:
Make 1mL solutions of BSA in water at 10, 8, 6, 4, 2, 1 µg/mL. I make 3mL of a 10µg/mL solution (1:1000 dilution of the BSA that comes with restriction enzymes) and then make each standard from this stock.

Method

Assay:
800µL protein sample (standards or unknown)
200µL Bradford reagent
• Mix by vortexing
• Don’t forget to do a blank (water instead of protein sample)
• Measure absorbance of standards and unknowns at 595nm (use 800+µL in plastic cuvettes)
• Unknowns may need to be significantly diluted in water; this assay’s dynamic range is only 0-10µg/mL.

Data analysis:
• Create a graph of protein concentration vs. absorbance for the BSA standards.
• Generate a linear regression line from the standards and obtain the line’s equation. R2 value should ideally be above 0.97.
• From the absorbances of the unknowns, solve the standard equation to find the protein concentration.
• Our new spectrophotometer can generate the regression for you automatically using your standards and will calculate the concentration for you.

Misc:
• You can get multiple uses without washing with one cuvette if you work from low to high concentration.
• If washing cuvettes for reuse, a rinse in dilute bleach solution is recommended to get the blue out.
• In the DU-7 spectrophotometer, you will get bad numbers if you use 600µL or less in the plastic cuvettes!! I recommend 800+µL in any spec.
• It’s advisable to make several different dilutions of your unknown sample. Ideally two or more of these will fall within the dynamic range of the assay and will yield similar results for undiluted concentration. I average all these.
• Keep in mind that all proteins react a little differently to this assay, so everything is relative to BSA.
• YES, YOU DO NEED TO RUN STANDARDS EVERY TIME! The reagent’s response will change even after one day.
• I am unaware of any component of our usual protein buffer solutions that interfere with this assay, but it doesn’t hurt to check. This assay is usually touted as being less sensitive than the Lowry to chemical interference.
• Easy conversion from µg/mL to µM: (µg/mL) * (1000) / (MW g/mol)