Bradford_Assay_Protocol - University of Illinois, Urbana Champaign MCB 253
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MCB 253
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MCB 253
Bradford Assay Protocol
Background:
The Bradford Assay technique is a quick and efficient way to determine the
concentration of a protein in a solution. The way it accomplishes this is to use a blue dye known
as Coomassie Brilliant Blue G-250 which when used within an acidic medium will change ...
bradford assay protocol background the bradford assay technique is a quick and efficient way to determine the concentration of a protein in a solution the way it accomplishes this is to use
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Benjamin Marsh
2/12/2021
MCB 253 Section Q
Bradford Assay Protocol
Background:
The Bradford Assay technique is a quick and efficient way to determine the
concentration of a protein in a solution. The way it accomplishes this is to use a blue dye known
as Coomassie Brilliant Blue G-250 which when used within an acidic medium will change the
color of the protein from brown to blue. According to Bio-Rad, the way it accomplishes this in an
acidic medium is "the dye is predominantly in the doubly protonated red cationic form”. When
the dye binds to protein, it is converted to an unprotonated form which is blue and has a
maximum absorbance at 595nm as the original absorbance was 470nm. As such using both
Beer’s Law and a spectrophotometer, the absorbance can be found at 595nm. Beer's Law is as
follows: A = ε * l * c, using this equation absorbance and concentration can easily be related.
The hypothesis for this experiment is we will use a Bradford assay to determine the
concentration of a stock solution of unknown protein. The BSA standard (2mg/mL) will be used
and 2mL of unknown protein to create a dilution scheme of 6 cuvettes each. The stocks will then
be mixed with these diluted protein samples with the Bradford reagent, allow it to incubate, and
finally place the samples in a Spectrophotometer to determine the absorbance at 595nm. If the
technique is done effectively then the proteins will fall under BSA standards.
Protocol:
1. Obtain 2 mL of BSA standard and the unknown protein solution each, 1.18 mL Bradford
reagent, and 20 μL buffer PBS.
2. Label 6 eppendorf tubes with the concentrations: 2 mg/mL (BSA 1), 1 mg/mL (BSA 2),
0.5 mg/mL (BSA 3), 0.25 mg/mL (BSA 4), 0.125 mg/mL (BSA 5), and 0.0625 mg/mL
(BSA 6).
a. These will be dilutions of the BSA standard.
3. Add 20 microliter PBS to BSA tubes 2, 3, 4, 5, and 6.
4. Using a 200 μL pipette, pipette 20 μL of the BSA from the 2 mg/mL tube (BSA 1), into the
1 mg/mL tube (BSA 2).
5. Repeat the same process, this time pipetting 20 μL from the 1 mg/mL tube into the 0.5
mg/mL tube. Repeat this process for 0.25, 0.125, and 0.0625 mg/mL tubes.
6. Mix tubes by vortexing with the micropipette by dispensing and releasing the plunger
repeatedly to mix the buffer and reagent.
a. Each eppendorf tube should have 20 μL total.
7. Label 6 eppendorf tubes as Protein 1, 2, 3, 4, 5, and 6. Add 20 μL PBS into Protein 2, 3,
4, 5, and 6.
a. These will be dilutions of unknown protein.
8. Using a 200 μL pipette, pipette 20 μL of the unknown protein from the 2 mg/μL tube
(Protein 1), into the 1 mg/μL tube (Protein 2).
9. Repeat the same process, this time pipetting 20 μL from Protein 2 into Protein 3. Repeat
this process for Protein 4, 5, and 6 tubes.
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