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
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)
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