User:Floriane Briere/Notebook/CHEM-496/2012/02/14: Difference between revisions
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==Objective== | ==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== | ==Protocol== | ||
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# Tube 3 (for 70 solution): 2.4mg 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 | * Tagging the peptides with the dye | ||
# Tube 1 (control solution): 160µL of dye + 10.32mL of control solution | # 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 2 (166 solution): 160µL of dye + 11.5mL of fibers solution | ||
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# Incubation for 24 hours at room temperature in the shaker | # 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== | |||
* Steven J. Bark and Klaus M. Hahn, Fluorescent Indicators of Peptide Cleavage in the Trafficking Compartments of Living Cells: Peptides Site-Specifically Labeled with Two Dyes, METHODS 20, 429–435 (2000) | |||
* "Dye labelling protocol": http://hahnlab.com/protocols.html | |||
* Dye characteristics: http://www.markergene.com/ProductDetails.php/M1259 | |||
Revision as of 11:41, 30 April 2012
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ObjectiveToday'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
ObservationsWhen we added the dye solution into our solutions, we observed an immediate color change.
References
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