The printable version is no longer supported and may have rendering errors. Please update your browser bookmarks and please use the default browser print function instead.
Chem-496
|
<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>
|
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)
- Control solution:
- Initial solution: pH = 3.98 and volume = 10mL
- Add 321µL of buffer
- Final solution: pH = 8.50 and volume = 10.32mL
- Fibers solution (BSA/Gold = 166):
- Initial solution: pH = 3.17 and volume = 10mL
- Add 1.5mL of buffer
- Final solution: pH = 8.51 and volume = 11.5mL
- Purple solution (BSA/Gold = 70):
- Initial solution: pH = 3.50 and volume = 10mL
- Add 600µL of buffer
- Final solution: pH = 8.50 and volume = 10.6mL
- Preparation of the dye solution
- Tube 1 (for control solution): 2.0mg of 5(6)-Carboxynaphthofluorescein, N-succinimidyl ester (473.51 g/mol) + 160µL of DMSO
- Tube 2 (for 166 solution): 2.0mg of 5(6)-Carboxynaphthofluorescein, N-succinimidyl ester (473.51 g/mol) + 160µL of DMSO
- 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
- Tube 1 (control solution): 160µL of dye + 10.32mL of control solution
- Tube 2 (166 solution): 160µL of dye + 11.5mL of fibers solution
- Tube 3 (70 solution): 160µL of dye + 10.6mL of purple solution
- 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
References
|