User:Alexander Cvitan/Notebook/Experimental Biological Chemistry Lab/2014/01/29

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Purpose

  • Make stock buffer solutions at specific pH's.
  • See the effect of the addition of these buffer solutions on Lysozyme AuNP fiber formation.

Procedure

  • Mix all test tubes in order to re-suspend the fibers in the solution. (Not to dissolve but to evenly distribute the fibers in the solution.)
  • Take 1 mL of solution from each test tube and place it in a separate 1.5 mL tube. (These aliquots will initially be used in order test each variable.)

Chelators

Creating Stock Solutions of Chelators

  • Note 2, 2 bipyridyl was replaced with 2, 2 bipyridine. The only difference between the two are the chirality is different. It still functions as chelator.

Adding Chelators to Lysozyme-AuNP Solutions

  • Add 0.5 mL of each chelator stock solution to each 1.5 mL aliquot.
  • Record effect of chelator on AuNP's.

pH

How we made each 1 M Buffer Solution

  • Please note that all of the following dilutions were calculated via an online buffer calculator found [HERE.]
Citric Acid: pH - 3
  • Add 42.02 g of citric acid in 180 mL distilled water.
  • Titrate to pH 3.0 with monovalent strong acid/base if necessary.
  • Fill up to 200 mL with distilled water.
Phosphate: pH - 7
  • Add 0.0908 mol of sodium phosphate monobasic to 0.1091 mol of sodium phosphate dibasic.
  • Fill up to 200 mL with distilled water.
Tris: pH - 9
  • Dissolve 24.228 g Tris base in 180 mL distilled water.
  • Titrate to pH 9.0 with monovalent strong acid/base if necessary.
  • Fill up to 200 mL with distilled water.
MES: pH - 5.21 (Note Cannot Get Down to 5)
  • Dissolve 39.04 g MES in 180 mL distilled water.
  • Titrate to pH 5.2 with monovalent strong acid/base if necessary.
  • Fill up to 200 mL with distilled water.
HEPES: pH - 7
  • Dissolve 47.66 g HEPES in 180 mL distilled water.
  • Titrate to pH 7 with monovalent strong acid/base if necessary.
  • Fill up to 200 mL with distilled water.
CHES: pH - 9
  • Dissolve 41.46 g CHES in 180 mL distilled water.
  • Titrate to pH 9 with monovalent strong acid/base if necessary.
  • Fill up to 200 mL with distilled water.

Adding Buffers to Lysozyme-AuNP Solutions

  • Dilute the 1 M solutions to 100 mM, 10 mM, and 1 mM.
  • Add 1 mL of each to the lysozyme-AuNP solutions.
  • Let sit overnight and record results.

Looking at the AuNP's

  • None of the 50:1 ratio AuNP's formed fibers. As a result they were remade because one of the stocks must have been slightly less concentrated.
  • Carly re-did the calculations and remade more 50:1 AuNP's. Nonetheless the chelators and and buffers were added to the other ratios made to see the effect. We will see the effect on the 50:1 ratio tomorrow.
  • Ratios 110:1 and 40:1 had chelator and buffer added to different aliquots. Their was no reaction immediately; however, solutions were left overnight.


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