Difference between revisions of "Bradford Assay"

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*BioRad Protein Assay Dye Reagent Concentrate cat#500-0006  
 
*BioRad Protein Assay Dye Reagent Concentrate cat#500-0006  
 
*Disposable Plastic Cuvette
 
*Disposable Plastic Cuvette
 +
*0.1mg/mL BSA in H20 as standard
  
 
==Protocol==
 
==Protocol==
 +
Cuvette Bradford Assay
 
#Dilute reagent 5X in water, stable for 2-3 weeks
 
#Dilute reagent 5X in water, stable for 2-3 weeks
 
#Pipet 1 mL into disposable plastic cuvette
 
#Pipet 1 mL into disposable plastic cuvette
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##Blank then measure samples, absorbance must be less than 0.9
 
##Blank then measure samples, absorbance must be less than 0.9
 
##Print (hit Recall, then enter, then print) and attach to experiment
 
##Print (hit Recall, then enter, then print) and attach to experiment
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 +
Protein Lysate Bradford Assay
 +
#Dilute reagent 5X in water, stable for 2-3 weeks
 +
#In a 96 well plate, dilute sample 20X (190ul H2O, 10ul Sample)--this dilution factor is tissue-dependent, only need to dilute fat ~5x
 +
#Add 5ul of 20x diluted sample to either 100ul or 200ul of Bradford reagent in well.
 +
##Prepare a standard curve by adding 0.0-3.0ul BSA std (1mg/ml) in increments of 0.5ul for 100 ul or 0-6ul BSA std in increments of 1ul for 200ul.
 +
#Run "Bradford Assay Protocol" on Plate Reader
  
 
==Reference==
 
==Reference==
 
*Wikipedia: [[wikipedia:Bradford_protein_assay|Bradford Protein Assay]]
 
*Wikipedia: [[wikipedia:Bradford_protein_assay|Bradford Protein Assay]]
 
*PMID 942051
 
*PMID 942051

Latest revision as of 14:39, 15 March 2018

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 either 100ul or 200ul of Bradford reagent in well.
    1. Prepare a standard curve by adding 0.0-3.0ul BSA std (1mg/ml) in increments of 0.5ul for 100 ul or 0-6ul BSA std in increments of 1ul for 200ul.
  4. Run "Bradford Assay Protocol" on Plate Reader

Reference

Retrieved from "http://bridgeslab.sph.umich.edu/protocols/index.php?title=Bradford_Assay&oldid=1461"