User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/02/03

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February 3rd, 2015

Analysis of last week...

Looks like only one of the two 57 μM samples prepared last week turned into a purple colloid solution. This was observed as early as Friday, although no specific measures have been taken to establish the exact length of time.

A plausible explanation for why the rest of the solutions failed to transform is the pH of the buffer solution used. Pepsin works optimally at an acidic pH of 1.5 - 3. The remainder of the proteases have optimal activity in the 7-8 pH range. As a result, we will repeat the same experiment with three additional proteases (trypsin, chymotrypsin, proteinase K) and also make a citric acid buffer to run the pepsin reaction in.

Solution preparation

1. A 115.6 μM stock solution of pepsin was made by dissolving 0.01625 g of pepsin in 4 mL of citric acid buffer.

  • From this stock solution, 4 samples of varying concentrations were prepared in the following manner:
    • 2.5 mL of stock + 2.5 mL of citric acid buffer --> P1 [pepsin]final = 57.8 μM
    • 0.5 mL of stock + 4.5 mL of citric acid buffer --> P2 [pepsin]final = 11.56 μM
    • 0.05 mL of stock + 4.95 mL of citric acid buffer --> P3 [pepsin]final = 1.156 μM
    • 0.005 mL of stock + 5.00 mL of citric acid buffer --> P4 [pepsin]final = 0.1156 μM


2. A 115.6 μM stock solution of trypsin was made by dissolving 0.01135 g of trypsin in 4 mL of Tris/NaCl buffer.

  • From this stock solution, 4 samples of varying concentrations were prepared in the following manner:
    • 2.5 mL of stock + 2.5 mL of Tris/Nacl buffer --> T1 [trypsin]final = 57.8 μM
    • 0.5 mL of stock + 4.5 mL of Tris/NaCl buffer --> T2 [trypsin]final = 11.56 μM
    • 0.05 mL of stock + 4.95 mL of Tris NaCl buffer --> T3 [trypsin]final = 1.156 μM
    • 0.005 mL of stock + 5.00 mL of Tris/NaCl buffer --> T4 [trypsin]final = 0.1156 μM


3. A 115.6 μM stock solution of chymotrypsin was made by dissolving 0.01170 g of chymotrypsin in 4 mL of Tris/NaCl buffer.

  • From this stock solution, 4 samples of varying concentrations were prepared in the following manner:
    • 2.5 mL of stock + 2.5 mL of Tris/Nacl buffer --> C1 [chymotrypsin]final = 57.8 μM
    • 0.5 mL of stock + 4.5 mL of Tris/NaCl buffer --> C2 [chymotrypsin]final = 11.56 μM
    • 0.05 mL of stock + 4.95 mL of Tris NaCl buffer --> C3 [chymotrypsin]final = 1.156 μM
    • 0.005 mL of stock + 5.00 mL of Tris/NaCl buffer --> C4 [chymotrypsin]final = 0.1156 μM


4. A 115.6 μM stock solution of proteinase K was made by dissolving 0.01345 g of proteinase K in 4 mL of Tris/NaCl buffer.

  • From this stock solution, 4 samples of varying concentrations were prepared in the following manner:
    • 2.5 mL of stock + 2.5 mL of Tris/Nacl buffer --> K1 [proteinase K]final = 57.8 μM
    • 0.5 mL of stock + 4.5 mL of Tris/NaCl buffer --> K2 [proteinase K]final = 11.56 μM
    • 0.05 mL of stock + 4.95 mL of Tris NaCl buffer --> K3 [proteinase K]final = 1.156 μM
    • 0.005 mL of stock + 5.00 mL of Tris/NaCl buffer --> K4 [proteinase K]final = 0.1156 μM

NOTE: Pepsin has an optimal pH of 1.5-2, so a pH 2.14, 5 mM Citrate, 0.2 M NaCl buffer was made for Pepsin use.

The AuNP fibers were spun down at 300 RPM for 4 minutes. The supernatant was removed, and the prepared solutions were added on top of the solid fibers. The tubes were placed in an incubator shaker at 37°C for analysis tomorrow.

Observations

Upon preparation of the 16 samples described above, each sample was manually shaken. All four samples of proteinase K demonstrated a "color change" from clear soln with solid AuNP fibers to purple colloid.


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