Difference between revisions of "Bradford Assay"
From Bridges Lab Protocols
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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 | ||
| Line 14: | Line 16: | ||
##Set dilution to be 1/vol (ie 0.1 for 10 uL) | ##Set dilution to be 1/vol (ie 0.1 for 10 uL) | ||
##Blank then measure samples, absorbance must be less than 0.9 | ##Blank then measure samples, absorbance must be less than 0.9 | ||
| − | ##Print and attach to experiment | + | ##Print (hit Recall, then enter, then print) and attach to experiment |
| + | |||
| + | 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]] | |
| + | *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
- Dilute reagent 5X in water, stable for 2-3 weeks
- Pipet 1 mL into disposable plastic cuvette
- Add 1-10 uL of protein sample, cover with parafilm and mix
- Let sit 5-10 min to react
- Set spectrophotometer as follows:
- Go to protein assay then Bradford assay
- Set formula, then select more
- Set b=0.045 (or determine slope)
- Set dilution to be 1/vol (ie 0.1 for 10 uL)
- Blank then measure samples, absorbance must be less than 0.9
- Print (hit Recall, then enter, then print) and attach to experiment
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
- Wikipedia: Bradford Protein Assay
- PMID 942051
