ADA Kinetics and more
Goals
- Run UV-Vis and AA on HRP/AuNPs samples
- Adenosine Deaminase Kinetics Assay
Procedure
- For procedure and data regarding the UV-Vis and the AA of the HRP and gold nanoparticle solutions, please see either Melissa's notebook or Dhea's notebook.
- The Adenosine Deaminase Kinetic assay was done by following the Assay Protocol
- The potassium Phosphate Buffer was actually sodium phosphate and was made at a pH of 7.4
- We made 50mL of .05M sodium phosphate buffer
.05 x .05= .0025 moles
.0025 moles x 268.07g/1mol = .6702g sodium phosphate in 50 mL H20. Actual amount = .6705. pH of 7.4 was achieve by adding roughly 3 drops of 12M HCl from a glass pasture pipette.
- 5mL of .1mM adenosine was made by adding 100mg of adenosine in 1mL of .05M phosphate buffer. This solution of adenosine was .00373M. .134mL of the .00373M adenosine solution was then diluted to a final volume of 5mL with .05M phosphate buffer giving a final concentration of .1mM adenosine.
- (0.001 g)/(268.02 g/mol) = 3.73×10-6 mol (3.73×10-6 mol)/0.001 L = 0.00373 M
(0.00373 M)(V1)=(5 mL)(0.1×10-3 M) V1 = 0.134 mL
- The UV-Vis was used to collect the spectras for the inosine, adenosine, and our purest fraction of ADA in buffer. The spectra peaks we were looking for were between the range of 200-300nm. For the inosine, 1mL of solution was placed in a quartz cuvet and the spectra was measured. All other samples had to be diluted because the spectras showed an absorbance of over 1. We initially added 1mL of adenosine followed by .5mL of adenosine and .5mL of sodium phosphate buffer. With the absorbance peaks still too high, we diluted the sample by a factor of 10 by placing 100μL adenosine in 900μL Buffer into the cuvet, however, this specra was still too high. As a result, we diluted the samples by a factor of 10 once more and in the final sample, there was 10μL adenosine and 990μL of buffer.
- For the ADA samples, used our purest most highly concentrated frac which is the fraction from november or newfrac. This was determined last week when we did the Bradford Assay. We initially started off by adding 15μL of ADA with 975μL of buffer. This spectra absorbance was once again too high and as a result, we decreased the amount of ADA present in the cuvet by a factor of 5. This resulted in the new amount of ADA in the cuvet to be 3μL with 997μL buffer. WIth this sample still being too high, we decided to do a serial dilution. 3μLof ADA and 997μL of buffer were diluted 4 times in the following manner. 500μL of solution and 500μL of buffer were mixed in a new cuvet. From that new solution in the cuvet, 500μL of new solution was mixed with 500μL of buffer in a new cuvet. This was done 4 times giving a final concentration of ADA in the cuvet to be 24.38nM.
65μM * 3μL= M2 * 1000μL
- dilution 1= .195μM
- dilution 2= 97.5nM
- dilution3= 48.7nM
- dilute 4= 24.38nM
Data
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