User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/10/08: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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*KI+AgNO<sub>3</sub>→AgI+AgNO<sub>3</sub>+KNO<sub>3</sub> | *KI+AgNO<sub>3</sub>→AgI+AgNO<sub>3</sub>+KNO<sub>3</sub> | ||
*AgNO<sub>3</sub>+NH<sub>4</sub>SCN→AgSCN+NH<sub>4</sub>NO<sub>3</sub> | *AgNO<sub>3</sub>+NH<sub>4</sub>SCN→AgSCN+NH<sub>4</sub>NO<sub>3</sub> | ||
**10. | **10.4 mL of NH<sub>4</sub>SCN were required to titrate excess AgNO<sub>3</sub>. (=1.00672×10<sup>-4</sup>mol) | ||
**2mL AgNO<sub>3</sub> contain 1.5×10<sup>-4</sup> mol | **2mL AgNO<sub>3</sub> contain 1.5×10<sup>-4</sup> mol | ||
**1.5×10<sup>-4</sup> | **1.5×10<sup>-4</sup>−1.00672×10<sup>-4</sup>=4.9328×10<sup>-5</sup> mol of AgNO<sub>3</sub> reacted with KI in a 1:1 reaction | ||
**1 mL of KI contains | **1 mL of KI contains 4.9328×10<sup>-5</sup> mol | ||
*'''[KI]= | *'''[KI]=49.328 mM''' | ||
*[I<sup>-</sup>] for dialysis | *[I<sup>-</sup>] for dialysis | ||
**'''Follow the same procedure as your KI standardization''' | **'''Follow the same procedure as your KI standardization''' | ||
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<u>Bradford Analysis</u> | <u>Bradford Analysis</u> | ||
*Only running bradford analysis of protein-containing solutions, that is Lys 1, 2, 3, 4 and 5, since the protein cannot diffuse through 3500 MWCO. | *Only running bradford analysis of protein-containing solutions, that is Lys 1, 2, 3, 4 and 5, since the protein cannot diffuse through 3500 MWCO. | ||
* Remove 20 μL of solution from each chamber ( | * Remove 20 μL of solution from each chamber (7 in all) and run Bradford analysis | ||
**Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl | **Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl | ||
**Recall, 20 μL solution + 200 μL diluted Bradford + 780 μL Tris/NaCl buffer | **Recall, 20 μL solution + 200 μL diluted Bradford + 780 μL Tris/NaCl buffer | ||
**PS cuvettes, measuring 400 - 800 nm | **PS cuvettes, measuring 400 - 800 nm | ||
** | [[Image:Bradford analysis 081014.png]] | ||
** | |||
<u>Bradford Calibration curve</u> | |||
*Did Bradford analysis for undialysed lysozyme solutions with concentrations 0.12, 0.3, 0.6 and 1 g/L. | |||
*The calibration curve below: | |||
[[Image:Lysozyme-Bradford CV.png]]<br.> | |||
<u>UV-Vis Absorption</u> <br.> | |||
Transfer 100 μL to a small volume UV cuvette & measure UV absorption | |||
**Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes | |||
**Measure entire 200 - 400 nm range. (Care about peak at 280 nm. Need to compare to fluorescence data to see how the binding is affected) | |||
<u>Fluorescence</u> | |||
*Measured fluorescence of dyalised solutions | |||
*Fluorescence (glass microcuvette & 100 fold diluted) for all protein solutions | |||
**10μL of lysozyme were diluted to 1mL by adding HPLC. (Remember to account for dilution factor later on) | |||
<u>[I-] titration</u> | |||
** AgNO<sub>3</sub> + NH<sub>4</sub>SCN → AgSCN + NH<sub>4</sub>NO<sub>3</sub> | |||
** KI + AgNO<sub>3</sub>(excess) → AgI (s) + AgNO<sub>3</sub> +KNO<sub>3</sub> | |||
*** Initial AgNO<sub>3</sub>: 0.075 M AgNO<sub>3</sub> x 0.002 L AgNO<sub>3</sub> = '''0.00015 mol AgNO<sub>3</sub>''' | |||
*** AgNO<sub>3</sub> (neutralized): 0.00968 M NH<sub>4</sub>SCN x 0.0104 L NH<sub>4</sub>SCN x (1 mol AgNO<sub>3</sub> / 1 mol NH<sub>4</sub>SCN ) = '''0.000100672 mol AgNO<sub>3</sub>''' | |||
*** Moles of KI: 0.00015 mol AgNO<sub>3</sub> <sub>initial</sub> - 0.000100672 mol AgNO<sub>3</sub> <sub>neutralized</sub> = '''0.0000493 mol KI''' | |||
*** [KI]= 0.0000493 mol KI / 0.001 L KI = '''49.3 mM KI''' | |||
Titrations were performed as follows: | |||
* 9.68 mM NH<sub>4</sub>SCN was titrated against 3 mL 1M HNO<sub>3</sub>, 10 mL deionized H<sub>2</sub>O , 200 μLFerric alum indicator, and 0.5 mL of dialyzed sample | |||
** Lysozyme against 2 mM KI | |||
*** 15.1 mL NH<sub>4</sub>SCN used | |||
** Lysozyme against 5 mM KI | |||
*** 14.2 mL NH<sub>4</sub>SCN used | |||
** Lysozyme against 10 mM KI | |||
*** 14.0 mL NH<sub>4</sub>SCN used | |||
** Lysozyme against 25 mM KI | |||
*** 13.7 mL NH<sub>4</sub>SCN used | |||
** Lysozyme against 50 mM KI | |||
*** 12.5 mL NH<sub>4</sub>SCN used | |||
** 2 mM KI against Lysozyme | |||
*** 13.6 mL NH<sub>4</sub>SCN used | |||
** 5 mM KI against Lysozyme | |||
*** 13.4 mL NH<sub>4</sub>SCN used | |||
** 10 mM KI against Lysozyme | |||
*** 13.2 mL NH<sub>4</sub>SCN used | |||
** 25 mM KI against Lysozyme | |||
*** 12.6 mL NH<sub>4</sub>SCN used | |||
** 50 mM KI against Lysozyme | |||
*** 11.9 mL NH<sub>4</sub>SCN used | |||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> |
Latest revision as of 00:23, 27 September 2017
Project name | Main project page Previous entry Next entry |
Tasks for October 8
[I-] by Titration PrecipitationProcedure followed as detailed by Dr. Fox
Fluorescence of protein solutions
Preparation of new Dialysis with 0.6 g/ L Lysozyme vs KI using 3500 MWCO
Dialysis DataBradford Analysis
Bradford Calibration curve
<br.> UV-Vis Absorption <br.> Transfer 100 μL to a small volume UV cuvette & measure UV absorption
Fluorescence
[I-] titration
Titrations were performed as follows:
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