User:Moira M. Esson/Notebook/CHEM-571/2013/09/10: Difference between revisions

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*Note: The UV-vis spectra was taken with the blank automatically subtracted out.
*Note: The UV-vis spectra was taken with the blank automatically subtracted out.
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'''Table 1.  
'''Table 1. Preparation of ADA standard solutions'''
 
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[[Image:Adenosine Deaminase (6) standard Table.png ]]
[[Image:Adenosine Deaminase (6) standard Table.png ]]
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Revision as of 07:57, 14 September 2013

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Objectives

  • Create two calibration curves for Adenosine Deaminase(ADA) using the Bradford assay technique.


Bradford Assay General Protocol

  • The following protocol was described here. (*Note: use section 2.3, page 5).
  1. Prepared a stock solution that is roughly 10mg ADA in 2mL buffer using a 2mL volumetric flask(Note the actual concentration prepared).
  2. Prepare 6 standard solutions (1μg/mL, 3μg/mL, 5μg/mL, 7μg/mL, 9μg/mL, 10μg/mL) in 1mL (prepared these solutions in an eppendorf tube.)
    1. Add 200μL of the Bio-Rad Protein Assay reagent to the eppendorf tube.
    2. Add necessary amount of protein from previously prepared 10mg in 2mL stock solution.
    3. Add necessary amount of buffer to fill eppendorf tube to 1mL.
    4. Shake the eppendorf tube vigourously for ~3min.
    5. Prepare 1mL of a blank solution (800μL buffer and 200μL Bio-Rad Protein Assay reagent.
    6. Take UV-vis spectra after preparation (no more than an hour after they were prepared).
    7. Prepare a calibration curve.
    8. Repeat Steps 1-7.
  • Note: All spectra were taken using a plastic cuvette.
  • Note: The UV-vis spectra was taken with the blank automatically subtracted out.


Table 1. Preparation of ADA standard solutions




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