Haynes:Bradford: Difference between revisions
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<- [ | <- [[Haynes:Protocols | Back to Protocols]] | ||
<div style="width: 800px"> | |||
=Bradford Assay= | |||
by Karmella Haynes, 2012<br><br> | |||
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 | ||
Principle: The dye in the Bradford reagent turns from brown to blue in the presence of protein. The | |||
Procedure | 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). | # 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 additional protein volume. | # 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. | ||
# 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). | # 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). | ||
# Add 5 μL of unknown to each remaining tube. Keep track of your samples with good labeling. | # Add 5.0 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 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 others, using new wells, but be sure to mix by pipetting up and down before transferring 200 μL of sample to the 96-well plate. | # Do the same for the others, using new wells, but be sure to mix by pipetting up and down before transferring 200 μL of sample to the 96-well plate. | ||
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# 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.<br>Protein concentration of the unknown = x μg/ '''5.0 μL'''. | ||
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Revision as of 16:34, 21 September 2013
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
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 additional protein volume.
- 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).
- Add 5.0 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 well in a clear 96-well flat-bottom plate.
- Do the same for the others, using new wells, but be sure to mix by pipetting up and down before transferring 200 μL of sample to the 96-well plate.
- Use a plate reader to record absorbance at 590 nm (OD 590).
What to do with your data: calculate unknown protein concentration(s)
- Subtract the blank OD 590 value 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.
Protein concentration of the unknown = x μg/ 5.0 μL.