TITLE: Bradford protein assay in 96-Well plate
AUTHORS: iGEM Dusseldorf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Assay for quantification of protein by comparing measured Absorbance at 595 nm to bovine serum albumine standard</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Bovine serum albumin (concentrated >200 µg/ml)</li><li style = "counter-reset:ol0;list-style-type:disc;">Roti-Quant 5x (ROTH)</li><li style = "counter-reset:ol0;list-style-type:disc;">96 Well Plate</li><li style = "counter-reset:ol0;list-style-type:disc;">Plate Reader</li></ul></div><div class = "text-block">Standard measurements</div><div class = "text-block">Dilute your BSA stock solution to 200µg/ml in the same buffer used for your solution of interest.</div><div class = "text-block">Dilute the Roti-Quant 5x reagent at a rate of 2:7,5. For 15 ml solution, this means 4 ml Roti-Quant added to 11 ml of Water.</div><div class = "text-block">Create 200 µl each of standard concentrations according to the following table:</div><table border><tr style = "text-align:center;"><td>  </td><td>A</td><td>B</td><td>C</td></tr><tr><td style = "text-align:center;">1</td><td rowspan = "" colspan = "" style ="display : table-cell;">Concentration [µg/ml]</td><td rowspan = "" colspan = "" style ="display : table-cell;">Volume of stock needed</td><td rowspan = "" colspan = "" style ="display : table-cell;">Volume of solution buffer needed</td></tr><tr><td style = "text-align:center;">2</td><td rowspan = "" colspan = "" style ="display : table-cell;">0</td><td rowspan = "" colspan = "" style ="display : table-cell;">0</td><td rowspan = "" colspan = "" style ="display : table-cell;">200</td></tr><tr><td style = "text-align:center;">3</td><td rowspan = "" colspan = "" style ="display : table-cell;">20</td><td rowspan = "" colspan = "" style ="display : table-cell;">20</td><td rowspan = "" colspan = "" style ="display : table-cell;">180</td></tr><tr><td style = "text-align:center;">4</td><td rowspan = "" colspan = "" style ="display : table-cell;">40</td><td rowspan = "" colspan = "" style ="display : table-cell;">40</td><td rowspan = "" colspan = "" style ="display : table-cell;">160</td></tr><tr><td style = "text-align:center;">5</td><td rowspan = "" colspan = "" style ="display : table-cell;">60</td><td rowspan = "" colspan = "" style ="display : table-cell;">60</td><td rowspan = "" colspan = "" style ="display : table-cell;">140</td></tr><tr><td style = "text-align:center;">6</td><td rowspan = "" colspan = "" style ="display : table-cell;">80</td><td rowspan = "" colspan = "" style ="display : table-cell;">80</td><td rowspan = "" colspan = "" style ="display : table-cell;">120</td></tr><tr><td style = "text-align:center;">7</td><td rowspan = "" colspan = "" style ="display : table-cell;">100</td><td rowspan = "" colspan = "" style ="display : table-cell;">100</td><td rowspan = "" colspan = "" style ="display : table-cell;">100</td></tr></table><div class = "text-block">Work in Triplicates. Pipette 50µl of the standards into the 96-Well plate</div><div class = "text-block">Add 200 µl of the diluted Bradford reagent to each well. </div><div class = "text-block">Incubate for 5 minutes at room temperature</div><div class = "text-block"><span>Measure the OD</span><span style = "vertical-align:sub;">595</span><span> for each well. Create a regression line from the averages of each concentrations' measurements</span></div><div class = "text-block">Protein concentration measurements</div><div class = "text-block">Dilute your solution of interest 1:20 or 1:40, depending on expected protein levels.</div><div class = "text-block">Work in triplicates and use blanks. Pipette 50 µl of each dilution into a well on the 96-Well plate.</div><div class = "text-block">When working with solutions containing pigments, prepare additional wells to which NO Bradford reagent will be added</div><div class = "text-block">Add 200 µl of the diluted Bradford reagent to each well.  For the pigment controls, add 200 µl of solution buffer instead. Optimally, use a multi-pipette to reduce the time needed for adding the reagent.</div><div class = "text-block">Incubate for 5 minutes at room temperature</div><div class = "text-block"><span>Measure the OD</span><span style = "vertical-align:sub;">595</span><span> for each well. </span></div><div class = "text-block"><span>When processing the data, correct the measurements for the blanks. When pigments are present in the solution, correct the OD</span><span style = "vertical-align:sub;">595</span><span> by subtracting the OD</span><span style = "vertical-align:sub;">595</span><span> of the pigment controls from the appropriate measurements.</span></div><div class = "text-block"><span>Determine the amount of protein by putting the achieved OD</span><span style = "vertical-align:sub;">595</span><span> of your solution of interest into the regression formula and multiplying by the dilution factor.</span></div></div>

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