User:Khyra A. Neal/Notebook/Chem 571/2014/09/10: Difference between revisions
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== | ==September 10, 2014== | ||
='''Bradford Assay'''= | |||
==Prepare Stock Solutions== | |||
*Prepare 50 mL of saline solution (0.9 wt-% NaCl) | |||
** 0.9 g→100 mL = 0.9 g/2= 0.45 g NaCl in 50 mL | |||
** Store in 45 mL falcon tube | |||
*Prepare 50 mL of 50 mM Tris base/ 50 mM NaCl solution | |||
**50 mM →1000 mL 2.5mM → 50 mL | |||
*** mass of tris= 2.5 mmol * 121.14 g/mol = 0.303 g Tris | |||
*** mass of NaCl= 2.5 mmol * 58.44 g/mol = 0.1461 g NaCl | |||
* Prepare stock solution of BSA 5mg in 5 mL →1.01 μg/μL | |||
** (0.00505 g BSA /mol) * (1 mol/66,430 g) / 0.005 L = 1.5204 X 10<sup>-5</sup>M = 0.0152 mM | |||
==UV-Vis Analysis== | |||
Record UV-Vis Spectrum for stock solutions (BSA, Saline Solution, and Tris Buffer) | |||
*Make 7 standard solutions (1mL each) of 1 μg/μL, 4μg/μL, 6μg/μL, 8μg/μL, 12μg/μL, 16μg/μL, and 20μg/mL | |||
**Volume of stock solution is determined based on the concentration chart below. | |||
==<font color=#0f1028></font>== | |||
{|style="width:300px" | |||
|<u>Concentration (μg/μL) </u> | |||
|<u>Amount of Bradford Reagent (μL)</u> | |||
|<u>Amount of Tris Buffer (μL)</u> | |||
|- | |||
|1 | |||
|200 | |||
|799 | |||
|- | |||
|4 | |||
|200 | |||
|796 | |||
|- | |||
|6 | |||
|200 | |||
|794 | |||
|- | |||
|8 | |||
|200 | |||
|792 | |||
|- | |||
|12 | |||
|200 | |||
|788 | |||
|- | |||
|16 | |||
|200 | |||
|784 | |||
|- | |||
|20 | |||
|200 | |||
|780 | |||
|} | |||
*All concentrations were diluted to 1 mL total volume with Tris buffer solution and placed in 1.5 mL centrifuge tube. | |||
*Tubes were closed and vortexed for 5-10 seconds and sat for 5 minutes before running UV-VIs. | |||
*Record UV-Vis Spectrum for stock solutions (BSA, Saline Solution, and Tris Buffer) between 200 nm and 800 nm. | |||
*Record UV-Vis Spectrum for 7 standard solutions between 400 nm and 800 nm. | |||
*Make duplicate blanks for | |||
**1 mL Tris/NaCl buffer | |||
**200 μL Bradford reagent + 800 μL buffer | |||
** Record their UV-Vis spectra between 400 nm and 800 nm. | |||
'''NOTE''' ALL UV-Vis spectra were run using polystyrene cuvettes. Solutions were discarded in waste bottle and polystyrene cuvettes were placed in a wash tube for cleaning. | |||
==Data Analysis== | |||
*Calculating the purity of BSA stock solution | |||
** | |||
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September 10, 2014Bradford AssayPrepare Stock Solutions
UV-Vis AnalysisRecord UV-Vis Spectrum for stock solutions (BSA, Saline Solution, and Tris Buffer)
NOTE ALL UV-Vis spectra were run using polystyrene cuvettes. Solutions were discarded in waste bottle and polystyrene cuvettes were placed in a wash tube for cleaning. Data Analysis
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