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

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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.
  3. Store any remaining ADA stock solution in the fridge.
  • 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)
1100.00560.794200
290.0050.795200
370.00390.7961200
450.00280.7972200
530.00170.7983200


  • 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)
0.01110.9889


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)
610.050.75200


UV/vis

Figure 1: Absorption spectra of ADA standard solutions Trial 1
Image:AbsorbanceADAbradford09102013zem.png
Figure 2. Absorption Spectra of ADA standard solutions Trial 2
Image:ADAtrial2chem57109102013zem.png]
Figure 3. Absorption Spectra of ADA standard solutions Trial 3
Image:ADAtrial309102013zem.png

UV/vis Notes

  • All three of the prepared graphs do not have to necessary shape of a Bradford assay, and on each graph, each of the concentrations have a different maximum absorption peak(most obvious in Figure 2.). This may be due to the fact that there is too small of a volume in the cuvettes. When a blank of water was placed in the cuvette with 1mL, there appeared to be extreme fluctuations. Another trial of the ADA bradford assay will be run tomorrow. Smaller cuvettes will be used.


AA Standard Preparations

  • Because AA is destructive 10mL of each sample will be prepared.
  • 5 standard solutions (25μg/mL, 20μg/mL, 15μg/mL, 10μg/mL, 5μg/mL) will be prepared.


General Protocol:

  1. Add the necessary volume of AA/ICPMS 1000 (±) 10 μL/mL, 10% HCl gold solution to a clean 10mL volumetric flask.
  2. Fill the volumetric flask to the line with deionized H2O. Cap and shake the volumetric flask for ~2min.
  3. Pour the gold solution into a falcon tube for storage. Parafilm the cap of the falcon tube to ensure no H2O evaporates.


Table 4. AA standard solutions preparation

Concentration(μg/mL) Amount Stock solution added (mL)
250.25
200.2
150.15
100.1
50.05





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