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

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==Objectives==
==Objectives==
* Create two calibration curves for Adenosine Deaminase(ADA) using the Bradford assay technique.   
* Create two calibration curves for Adenosine Deaminase(ADA) using the Bradford assay technique.   
* Prepare 5 standard gold solutions for AA testing
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==Bradford Assay General Protocol==
==Bradford Assay General Protocol==
*The following protocol was described [http://www.bio-rad.com/webroot/web/pdf/lsr/literature/LIT33.pdf here]. (*Note: use section 2.3, page 5).
*The following protocol was described [http://www.bio-rad.com/webroot/web/pdf/lsr/literature/LIT33.pdf here]. (*Note: use section 2.3, page 5).

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Objectives

  • Create two calibration curves for Adenosine Deaminase(ADA) using the Bradford assay technique.
  • Prepare 5 standard gold solutions for AA testing


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 from 10mg/2mL stock solution

Sample Number Concentration (μg/mL) ADA (mL) Buffer (mL) Coomassive Blue(μL)
1 10 0.0056 0.794 200
2 9 0.005 0.795 200
3 7 0.0039 0.7961 200
4 5 0.0028 0.7972 200
5 3 0.0017 0.7983 200


  • Note: 1μg/mL was too small of a volume to prepare from the 10mg/mL stock. Another stock solution of 20μg/μL was prepared(with buffer and ADA from the stock solution) and a dilution to 1μg/μL was performed from the 20μg/μL solution.


Table 2. Preparation of 20μg/μL ADA standard solution

ADA (mL) Buffer(mL)
0.0111 0.9889


Table 3. Preparation of 1μg/μL ADA standard solution from 20μg/μL ADA standard solution

Sample number Concentration(μg/μL) ADA (mL) Buffer(mL) Coomassive Blue(μL)
6 1 0.05 0.75 200





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