Team:Yale/LabNotebook/Cu

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(Difference between revisions)
(→Oct 12, 2010; Standard curve troubleshooting)
(→Oct 12, 2010; Standard curve troubleshooting)
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[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T38-41J66VR-B&_user=483702&_coverDate=11/01/2000&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_rerunOrigin=scholar.google&_acct=C000022720&_version=1&_urlVersion=0&_userid=483702&md5=4dfd3ba70e1e7ab05c0fb6950583baed&searchtype=a#secx9 Lebeau et al, 2000] used this method:
[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T38-41J66VR-B&_user=483702&_coverDate=11/01/2000&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_rerunOrigin=scholar.google&_acct=C000022720&_version=1&_urlVersion=0&_userid=483702&md5=4dfd3ba70e1e7ab05c0fb6950583baed&searchtype=a#secx9 Lebeau et al, 2000] used this method:
*Measurement of Cu(II) reduction.  Seven hundred seventy μl of BDA stock solution (5.7 mM) BDA in 5.2 M ammonium acetate/10% ethanol was mixed with 720 μL of CuSO4 solution (1.25 mM) in water and 10 μL of test compounds (15 mM) except quercetin (1.5 mM) dissolved in ethanol [37]. The complex formation (BDA/Cu(I)) was followed by optical density measurements made at 484 nm. The amount of reduced copper was determined from a calibration curve constructed using solutions of Cu(II) (0–300 μM) reduced by ascorbate (1 mM) in the presence of BDA (2.7 mM). The percent of ethanol in water for the dosage does not exceed 5.6%.
*Measurement of Cu(II) reduction.  Seven hundred seventy μl of BDA stock solution (5.7 mM) BDA in 5.2 M ammonium acetate/10% ethanol was mixed with 720 μL of CuSO4 solution (1.25 mM) in water and 10 μL of test compounds (15 mM) except quercetin (1.5 mM) dissolved in ethanol [37]. The complex formation (BDA/Cu(I)) was followed by optical density measurements made at 484 nm. The amount of reduced copper was determined from a calibration curve constructed using solutions of Cu(II) (0–300 μM) reduced by ascorbate (1 mM) in the presence of BDA (2.7 mM). The percent of ethanol in water for the dosage does not exceed 5.6%.
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[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4WBK7DP-4&_user=483702&_coverDate=09/01/2009&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000022720&_version=1&_urlVersion=0&_userid=483702&md5=98048357a77f175010bf42a5d109fdf4&searchtype=a#bbib25 Campos et al 2009] used this method:
 +
*Different concentrations of copper sulfate (0.05–0.8 mM) and BCS (0.1–1.6 mM) were investigated by altering one variable at a time to optimize the assay. Final concentrations of 0.2 mM BCS and 0.1 mM CuSO4 gave enough absorption intensity at 490 nm with good linearity up to 2 mM trolox, as is shown below; therefore, they were established as optimal for the assay. EDTA (1.67 mM final concentration) is used as a stop reagent. When EDTA is added at this concentration before the sample, no color is developed (Fig. 1). On the other hand, we noticed that when a concentration of EDTA higher than 1.67 mM is used, a precipitate could appear in plasma samples.
===Useful links===
===Useful links===

Revision as of 17:28, 12 October 2010

General description of goal of this part of the project.

Contents


Oct 06, 2010; Making stock solutions

Generated the stock solutions for the Followed the standard curve protocol.

  • Made a 100 mM stock solution of BCS.
  • Made two CuSO4 dilution series, in LB and MM, from 10 mM Cu to 10 pM.

Oct 07, 2010; Bacterial growth assays, Cu ppt

# Sample ID Date and Time 1 (nm) 1 (Abs)
1o/n culture10/7/2010 11:16:10 AM6000.854
2MM t010/7/2010 11:44:45 AM6000.002
3LB t010/7/2010 11:31:06 AM6000.089
4LB t110/7/2010 12:47:55 PM6000.189
5MM t110/7/2010 12:45:13 PM6000.002
6MM t210/7/2010 1:41:51 PM6000.004
7LB t210/7/2010 1:43:38 PM6000.450
8MM t310/7/2010 2:44:50 PM6000.001
9LB t310/7/2010 2:46:09 PM6000.761
10MM t410/7/2010 3:53:12 PM6000.005
11LB t410/7/2010 3:54:36 PM6001.064

Oct 12, 2010; Standard curve troubleshooting

[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T38-41J66VR-B&_user=483702&_coverDate=11/01/2000&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_rerunOrigin=scholar.google&_acct=C000022720&_version=1&_urlVersion=0&_userid=483702&md5=4dfd3ba70e1e7ab05c0fb6950583baed&searchtype=a#secx9 Lebeau et al, 2000] used this method:

  • Measurement of Cu(II) reduction. Seven hundred seventy μl of BDA stock solution (5.7 mM) BDA in 5.2 M ammonium acetate/10% ethanol was mixed with 720 μL of CuSO4 solution (1.25 mM) in water and 10 μL of test compounds (15 mM) except quercetin (1.5 mM) dissolved in ethanol [37]. The complex formation (BDA/Cu(I)) was followed by optical density measurements made at 484 nm. The amount of reduced copper was determined from a calibration curve constructed using solutions of Cu(II) (0–300 μM) reduced by ascorbate (1 mM) in the presence of BDA (2.7 mM). The percent of ethanol in water for the dosage does not exceed 5.6%.

[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4WBK7DP-4&_user=483702&_coverDate=09/01/2009&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000022720&_version=1&_urlVersion=0&_userid=483702&md5=98048357a77f175010bf42a5d109fdf4&searchtype=a#bbib25 Campos et al 2009] used this method:

  • Different concentrations of copper sulfate (0.05–0.8 mM) and BCS (0.1–1.6 mM) were investigated by altering one variable at a time to optimize the assay. Final concentrations of 0.2 mM BCS and 0.1 mM CuSO4 gave enough absorption intensity at 490 nm with good linearity up to 2 mM trolox, as is shown below; therefore, they were established as optimal for the assay. EDTA (1.67 mM final concentration) is used as a stop reagent. When EDTA is added at this concentration before the sample, no color is developed (Fig. 1). On the other hand, we noticed that when a concentration of EDTA higher than 1.67 mM is used, a precipitate could appear in plasma samples.

Useful links

LabNotebook

[http://meta.wikimedia.org/wiki/Help:Wikitext_examples Wiki formatting help]

[http://excel2wiki.net/ Excel table to Wiki format tool]

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