User:Khyra A. Neal/Notebook/Chem 571/2014/09/10: Difference between revisions

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==Entry title==
==September 10, 2014==
* Insert content here...
='''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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Revision as of 22:50, 16 September 2014

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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 10-5M = 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.

Concentration (μg/μL) Amount of Bradford Reagent (μL) Amount of Tris Buffer (μL)
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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