Bradford Assay

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The third part of the experiment utilized the Bradford Assay which helped determine the concentration of the unknown protein samples using measures that helped in forming a standard curve, after conducting gel electrophoresis. These calculated amounts are seen in Table 1 and were taken from a previous experiment (Spectroscopy, 2015). Table 1- Calculated Amounts for Standard Curve µg of BSA needed Volume of BSA (µL) Volume of H2O (µL) Volume of Bradford Reagent (µL) Total volume of mixture (µL) Final concentration BLANK ----- 950 µL 50 µL 1000 µL ----- 2.5 µg 5 945 µL 50 µL 1000 µL 0.0025 5 µg 10 940 µL 50 µL 1000 µL 0.005 10 µg 20 930 µL 50 µL 1000 µL 0.01 20 µg 40 910 µL 50 µL 1000 µL 0.02 Once the final concentrations were determined, a…show more content…
Figure 1- The Standard Curve Using BSA From the standard curve, the concentrations of the unknown protein samples P1 (0.451 A) and P2 (0.373 A) were determined: P1: 0.451=16.922x+0.1006 0.3504/16.922=x=0.021…show more content…
Added 0.50 mL of the protein mix into the column and collected the contents in an Eppendorf tube (label it P1). Washed the column 2 times with 0.50 mL of diluted water, and discarded the flow-through. Added 0.50 mL of 50 mM Tris buffer (8.8 pH) which has 50 mM of NaCl; labeled the contents in a second Eppendorf tube as P2. Part 2 – SDS-PAGE Prepared the gel to load both protein samples, P1 and P2. Added 10 µL of the loading dye to each Eppendorf tube. Transferred 5 µL of each protein sample in 2 other tubes, mixed them, and boiled them for about 2 minutes. 10 µL of the molecular weight ladder was loaded in the first lane of the gel, and then the other protein samples were loaded in the rows following. Ran the gel at 150 V. Took out the gel and stained it with Coomassie for about 5-10 minutes. Washed the gel with diluted water until the water came out as clear. Destained the gel with water, in the microwave for about 5 minutes. Part 3 – Measuring the Concentration of P1 and P2 Prepared BSA (bovine serum albumin) for a standard curve, similar to the table from the Spectroscopy Lab. Has to be done every time one has to determine the concentration of a
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