Haynes:Bradford: Difference between revisions
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<- [Back | <- [[Haynes:Protocols | Back to Protocols]] | ||
<div style="width: 800px"> | |||
=Bradford Assay= | |||
by Karmella Haynes, 2012<br><br> | |||
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 | |||
# Label enough 1.5 mL eppendorf tubes for one blank (1) , five standard samples (2-6), and all of your unknown samples (7-n). | * BioTek Synergy H1 Plate Reader (or similar) | ||
# Add 500 μL Bradford Reagent to each tube. You will add protein to these later, and ignore the negligible change caused by additional | |||
# Add a BSA standard protein solution | |||
# Add 5 μL of unknown to each remaining tube. Keep track of your samples with good labeling. | 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. | # 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 | # Do the same for the other samples, using new wells. | ||
# Use a plate reader to record absorbance at 590 nm (OD 590). | # 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) | What to do with your data: calculate unknown protein concentration(s) | ||
# Subtract the blank OD 590 value from all other values. | # 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]. | # 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. | # 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. | # 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 | |||
</div> |
Latest revision as of 13:36, 15 September 2014
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
- 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 dH2O 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
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)
- 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 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