User:Floriane Briere/Notebook/CHEM-496/2012/02/14

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Contents

Objective

Today's objective is to tag our BSA proteins with our dye. The dye we are using is 5(6)-Carboxynaphthofluorescein, N-succinimidyl ester and has been obtained from Marker Gene Technologies Inc. This dye is able to tag unprotonated lysine residues; for this reason the first step of our protocol is to increase the pH of our solutions (BSA/HCl control, 70 ratio, 166 ratio) so that it reaches a pH of 8.5 (because the lysine pKa = 8.95 for the amino group); to do so, we are going to add some buffer (prepared on 8/2/12) to the solution until the pH reaches 8.5 (we are checking the pH using a SB70P pHmeter).

Then, because the dye isn't water soluble, we have to prepare a satured DMSO solution; we are going to dissolve the dye (whose mass has been determined on 8/2/12) into as few DMSO as possible. To finish, we'll leave the reaction occur overnight; the next step will be to perform a dialysis to remove the excess of dye.

Protocol

  • Changing the pH of the solution (to 8.50)
  1. Control solution:
    1. Initial solution: pH = 3.98 and volume = 10mL
    2. Add 321µL of buffer
    3. Final solution: pH = 8.50 and volume = 10.32mL
  2. Fibers solution (BSA/Gold = 166):
    1. Initial solution: pH = 3.17 and volume = 10mL
    2. Add 1.5mL of buffer
    3. Final solution: pH = 8.51 and volume = 11.5mL
  3. Purple solution (BSA/Gold = 70):
    1. Initial solution: pH = 3.50 and volume = 10mL
    2. Add 600µL of buffer
    3. Final solution: pH = 8.50 and volume = 10.6mL
  • Preparation of the dye solution
  1. Tube 1 (for control solution): 2.0mg of 5(6)-Carboxynaphthofluorescein, N-succinimidyl ester (473.51 g/mol) + 160µL of DMSO
  2. Tube 2 (for 166 solution): 2.0mg of 5(6)-Carboxynaphthofluorescein, N-succinimidyl ester (473.51 g/mol) + 160µL of DMSO
  3. Tube 3 (for 70 solution): 2.4mg of 5(6)-Carboxynaphthofluorescein, N-succinimidyl ester (473.51 g/mol) + 160µL of DMSO
  • Tagging the peptides with the dye
  1. Tube 1 (control solution): 160µL of dye + 10.32mL of control solution
  2. Tube 2 (166 solution): 160µL of dye + 11.5mL of fibers solution
  3. Tube 3 (70 solution): 160µL of dye + 10.6mL of purple solution
  4. Incubation for 24 hours at room temperature in the shaker

Observations

When we added the dye solution into our solutions, we observed an immediate color change.

  • For the control solution (tube 1), it was initially a clear solution; when we added the dye we observed the formation of a gradient (from purple to blue)
  • For the fibers solution (tube 2), it was initially a clear solution with some purple fibers in it; when we added the dye the solution turned blue
  • For the purple solutoin (tube 3), it was initially a light purple solution; when we added the dye the solution turned dark purple

Image:14fev-Tagging BSA.jpg

References

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