Difference between revisions of "Bradford Assay"

Revision as of 15:52, 8 August 2017

Materials

• BioRad Protein Assay Dye Reagent Concentrate cat#500-0006
• Disposable Plastic Cuvette
• 0.1mg/mL BSA in H20 as standard

Protocol

Cuvette Bradford Assay

1. Dilute reagent 5X in water, stable for 2-3 weeks
2. Pipet 1 mL into disposable plastic cuvette
3. Add 1-10 uL of protein sample, cover with parafilm and mix
4. Let sit 5-10 min to react
5. Set spectrophotometer as follows:
   1. Go to protein assay then Bradford assay
   2. Set formula, then select more
   3. Set b=0.045 (or determine slope)
   4. Set dilution to be 1/vol (ie 0.1 for 10 uL)
   5. Blank then measure samples, absorbance must be less than 0.9
   6. Print (hit Recall, then enter, then print) and attach to experiment

Protein Lysate Bradford Assay

1. Dilute reagent 5X in water, stable for 2-3 weeks
2. In a 96 well plate, dilute sample 20X (190ul H2O, 10ul Sample)--this dilution factor is tissue-dependent, only need to dilute fat ~5x
3. Add 5ul of 20x diluted sample to 100ul of reagent in well
4. Run "Bradford Assay Protocol" on Plate Reader

Reference

• Wikipedia: Bradford Protein Assay
• PMID 942051