User:Omar Choudary/Notebook/Omar Choudary CHEM-571 AU-2011/2012/2012/02/07

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Contents

Objective

  • Prepare control solution of BSA in H2O
  • Determine how to produce Tris refolding buffer for dyeing Au/BSA mixture.
  • Determine how much Dye ( 5(6)-Carboxynaphthofluorescein, N-Succinimidyl Ester ) to add to Au/BSA mixture in refolding buffer to successfully tag lysine residues.

Description

  • 1 mL of 17.7 μM BSA was added to 9 mL H2O in test tube.
  • Solution was placed in oven set at 80 degrees Celsius for 2 hours and then removed.

Data

TRIS BUFFER CALCULATIONS:

  • Stock Solutions : .948 M HCl , 13.3 mM Tris buffer
  • 8.5 pH Tris = 8.06 + log [Salt] / [Acid]

[Salt] / [Acid] = 2.754

  • (50 - x) / (x) = 2.754

x = 13.3 mM Tris Buffer

  • (0.133 M Tris) * ( .25 L ) = (0.948 M HCl) * (x L)

x = 3.51 mL HCl

  • (0.050 M Tris) * (0.25 L) * ( 121.14 g / mole Tris) = 1.514 g Tris


DYE CALCULATIONS:

  • Stock solution of 17.7 μM BSA:
  • (17.7 * 10 ^ -6 / L ) * (1 * 10 ^ -3) = 1.77 * 10 ^ -8 moles BSA
  • 1.77 * 10 ^ -8 moles BSA = x / 6.022 * 10 ^ 23 molecules
  • x = 1.0659 x 10 ^ 16 molecules of BSA
  • BSA contains 60 lysine residues per molecule [1]
  • 60 lysine residues * 1.0659 * 10 ^ 16 molecules of BSA = 6.395 * 10 ^ 17 lysine residues
  • Dye ( 5(6)-Carboxynaphthofluorescein, N-Succinimidyl Ester ) Molecular Weight = 573.51 grams/mole
  • For 3x excess dye, ( 3 * 6.395 * 10 ^ 17 lysine residues) = 1.9185 * 10 ^ 18 lysine residues
  • 1.9185 * 10 ^ 18 lysine residues / 6.022 * 10 ^ 23 molecules = 3.1858 * 10 ^ -6 moles Dye
  • 3.1858 * 10 ^ -6 moles * (573.51 grams / 1 mole Dye) = 1.827 * 10 ^ -3 grams of Dye (1.827 mg Dye)

Notes

  • The BSA control solution was placed in the oven to facilitate unfolding of the protein, but it's possible that removal from the oven will facilitate refolding.
  • Another control will be made using 1 mL of the BSA solution, 1 mL of HCl, and 8 mL of H2O to reduce the potential of the BSA to refold.
  • Dye Selected: 5(6)-Carboxynaphthofluorescein, N-succinimidyl ester ( C33H19NO9) – Marker Gene Technologies Inc.

-Absorption: 602 nm, Emission: 672 nm

-Reactive long-wave fluorophore

-Reacts with terminal and internal Lysine residues

-pH dependent (8.0 - 9.0 range)


Image:2-7-2012-1.jpg

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