Haynes:Bradford: Difference between revisions

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(New page: '''Bradford Assay'''<br> by Karmella Haynes, 2012 Principle: The dye in the Bradford reagent turns from brown to blue in the presence of protein. The absorbance f blue color is proportion...)
 
 
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'''Bradford Assay'''<br>
<- [[Haynes:Protocols | Back to Protocols]]
by Karmella Haynes, 2012


Principle: The dye in the Bradford reagent turns from brown to blue in the presence of protein. The absorbance f blue color is proportional to the protein concentration. See http://en.wikipedia.org/wiki/Bradford_protein_assay
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# Label enough 1.5 mL eppendorf tubes for one blank (1) , five standard samples (2-6), and all of your unknown samples (7-n).
=Bradford Assay=
# Add 500 μL Bradford Reagent to each tube. You will add protein to these later, and ignore the negligible change caused by additional protein volume.
by Karmella Haynes, 2012<br><br>
# Add a BSA standard protein solution* to tubes 2 (1μg BSA), 3 (2μg BSA), 4 (4μg BSA), 5 (8μg BSA), and 6 (16μg BSA). (*Note, use the appropriate volume based on the concentration of your stock BSA).
Principle: The dye in the Bradford reagent turns from brown to blue in the presence of protein. The color change is proportional to the protein concentration. See http://en.wikipedia.org/wiki/Bradford_protein_assay
# Add 5 μL of unknown to each remaining tube. Keep track of your samples with good labeling.
 
# Transfer 200 μL of the blank (tube one) into the first del in a clear 96-well flat-bottom plate.
MATERIALS
* Transparent flat-bottom 96-well plate (e.g. Greiner Bio-One 655101)
* Bradford reagent (e.g., Sigma B6916-500ML)
* Bovine Serum Albumin (BSA) (e.g., New England Biolabs 10 mg/mL B9001S)
 
 
EQUIPMENT
* BioTek Synergy H1 Plate Reader (or similar)
 
 
PROCEDURE
# Label enough 1.5 mL eppendorf tubes for one blank (1) , five standard samples (2-6), and all of your unknown samples (7-...''n'').
# Add 500 μL Bradford Reagent to each tube. You will add protein to these later, and ignore the negligible change caused by the additional volume.
# Dilute the stock BSA in a new tube to make 50 μL of 1 μg/μL BSA. Example: if the stock BSA is 10 mg/mL, add 5 μL of BSA to 45 μL dH<sub>2</sub>O in a fresh tube.
# Add a BSA standard protein solution to tubes 1-6. See '''Table 1'''.
# Add 5.0 μL of unknown to each remaining tube. Keep track of your samples with good labeling!
# Close all caps and invert the tubes to thoroughly mix the samples.
# Transfer 200 μL of the blank (tube one) into the first well in a clear 96-well flat-bottom plate.
# Do the same for the other samples, using new wells.
# Use a plate reader to record absorbance at 590 nm (OD 590). If using the BioTek Synergy H1 Software, set up a new protocol and under Procedure > Action > Read use the following settings
## Detection Method = Absorbance
## Read Type = Endpoint
## Wavelength (1) = 590 nm (type-in the value manually)
 
 
'''Table 1. Standard sample set-up'''
{| {{table}}
|-
| Reagent || Tube 1<br>(0 μg BSA) || Tube 2<br>(1 μg BSA) || Tube 3<br>(2 μg BSA) || Tube 4<br>(4 μg BSA) || Tube 5 <br>8 μg BSA) || Tube 5<br>(16 μg BSA)
|-
| Bradford Reagent || 500 μL || 500 μL || 500 μL || 500 μL || 500 μL || 500 μL
|-
| BSA diluted to 1 μg/μL || 0 μL || 1.0 μL || 2.0 μL || 4.0 μL || 8.0 μL || 16.0 μL
|}
 
 
What to do with your data: calculate unknown protein concentration(s)
# Subtract the blank OD 590 value (Tube 1) from all other values.
# Plot a standard curve (using Excel) with BSA concentration (x-axis) vs. Absorbance at 590 nm (y-axis). See [http://openwetware.org/wiki/Image:KAH_012510_chart1.tif this example].
# Add a '''line''' of best fit (not a curve) and display the equation.
# Solve the equation for ''x''. Substitute ''y'' with the background-subtracted OD 590 for the unknowns. The x value will be the protein concentration of the unknown as μg/'''5.0 μL''' (because you used 5 μL to set up the assay sample).
# Convert the unknowns to μg/μL: (x μg/5.0 μL) / 5 = x μg/μL
 
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Latest revision as of 13:36, 15 September 2014

<- Back to Protocols

Bradford Assay

by Karmella Haynes, 2012

Principle: The dye in the Bradford reagent turns from brown to blue in the presence of protein. The color change is proportional to the protein concentration. See http://en.wikipedia.org/wiki/Bradford_protein_assay

MATERIALS

  • Transparent flat-bottom 96-well plate (e.g. Greiner Bio-One 655101)
  • Bradford reagent (e.g., Sigma B6916-500ML)
  • Bovine Serum Albumin (BSA) (e.g., New England Biolabs 10 mg/mL B9001S)


EQUIPMENT

  • BioTek Synergy H1 Plate Reader (or similar)


PROCEDURE

  1. Label enough 1.5 mL eppendorf tubes for one blank (1) , five standard samples (2-6), and all of your unknown samples (7-...n).
  2. Add 500 μL Bradford Reagent to each tube. You will add protein to these later, and ignore the negligible change caused by the additional volume.
  3. Dilute the stock BSA in a new tube to make 50 μL of 1 μg/μL BSA. Example: if the stock BSA is 10 mg/mL, add 5 μL of BSA to 45 μL dH2O in a fresh tube.
  4. Add a BSA standard protein solution to tubes 1-6. See Table 1.
  5. Add 5.0 μL of unknown to each remaining tube. Keep track of your samples with good labeling!
  6. Close all caps and invert the tubes to thoroughly mix the samples.
  7. Transfer 200 μL of the blank (tube one) into the first well in a clear 96-well flat-bottom plate.
  8. Do the same for the other samples, using new wells.
  9. Use a plate reader to record absorbance at 590 nm (OD 590). If using the BioTek Synergy H1 Software, set up a new protocol and under Procedure > Action > Read use the following settings
    1. Detection Method = Absorbance
    2. Read Type = Endpoint
    3. Wavelength (1) = 590 nm (type-in the value manually)


Table 1. Standard sample set-up

Reagent Tube 1
(0 μg BSA)		 Tube 2
(1 μg BSA)		 Tube 3
(2 μg BSA)		 Tube 4
(4 μg BSA)		 Tube 5
8 μg BSA)		 Tube 5
(16 μg BSA)
Bradford Reagent 500 μL 500 μL 500 μL 500 μL 500 μL 500 μL
BSA diluted to 1 μg/μL 0 μL 1.0 μL 2.0 μL 4.0 μL 8.0 μL 16.0 μL


What to do with your data: calculate unknown protein concentration(s)

  1. Subtract the blank OD 590 value (Tube 1) from all other values.
  2. Plot a standard curve (using Excel) with BSA concentration (x-axis) vs. Absorbance at 590 nm (y-axis). See this example.
  3. Add a line of best fit (not a curve) and display the equation.
  4. Solve the equation for x. Substitute y with the background-subtracted OD 590 for the unknowns. The x value will be the protein concentration of the unknown as μg/5.0 μL (because you used 5 μL to set up the assay sample).
  5. Convert the unknowns to μg/μL: (x μg/5.0 μL) / 5 = x μg/μL
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