User:Khyra A. Neal/Notebook/Chem 571/2014/10/29: 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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[[Image:Bradford_of_Lys_vs_CaCl2.jpg]] | [[Image:Bradford_of_Lys_vs_CaCl2.jpg]] | ||
3. Ca<sup>2+</sup> | 3. | ||
<u>Ca<sup>2+</sup> ISE</u> | |||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''CaCl<sub>2</sub> concentration''' | | align="center" style="background:#f0f0f0;"|'''CaCl<sub>2</sub> concentration''' | ||
| Line 61: | Line 60: | ||
* Use SDS, HCl, HPLC, & methanol to clean cuvette after each use | * Use SDS, HCl, HPLC, & methanol to clean cuvette after each use | ||
* Fluorescence spectra was obtained to determine how binding of the protein was affected | * Fluorescence spectra was obtained to determine how binding of the protein was affected | ||
[[Image:Fluorescence_Lys_vs_CaCl2.jpg]] | |||
[[Image:UV_Lys_vs_CaCl2.jpg]] | |||
'''NOTE''' Both the absorption spectra and emission spectra are indicative that an equilibrium is reached with 42 μM Lysozyme and concentrations below 5 mM CaCl<sub>2</sub>. The protein concentration reaches at max absorbance at 500 μM CaCl<sub>2</sub> and then decreases at 5 mM and 50 mM, respectively, indicating that lysozyme is precipitating out of the solution. | |||
6. Solution Preparation | |||
* Prepare new 0.6 g/L Lysozyme | |||
** '''0.0380 g''' of lysozyme dissolved in 50 mL H<sub>2</sub>O | |||
* Prepare 5 mM Stock HCl solution | |||
** Add '''0.5 mL''' of 0.5 M HCL to 50 mL distilled H<sub>2</sub>O | |||
*** Dilutions were prepared from 5 mM HCl stock solution | |||
**** 75 μM, 100 μM, 250 μM, and 500 μM | |||
7. Prepare New Dialysis | |||
* 42 μM Lysozyme (0.6 g/L) vs HCl | |||
** Use 20,000 MWCO tubing | |||
** Add 1 mL 0.6 g/L Lysozyme to 5 cells on one side of chamber | |||
** Add 1 mL 75 μM, 100 μM, 250 μM, 500 μM, and 5 mM HCl to the opposite side of the chamber (one concentration per cell) | |||
** Secure wells by screwing them to prevent any evaporation | |||
** Place on low speed shaker for one week and prepare for analysis | |||
__NOTOC__ | __NOTOC__ | ||
Latest revision as of 00:29, 27 September 2017
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October 29, 2014Tasklist1. Extract Lysozyme and CaCl2 solutions
2. Bradford Analysis
3. Ca2+ ISE
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5. UV-Vis and Fluorescence
- Sample Solutions were diluted 1:100
- Transfer 100 μL of sample solution to a small volume cuvette
- Measure absorbance between 200 nm and 800 nm
- Use SDS, HCl, HPLC, & methanol to clean cuvette after each use
- Fluorescence spectra was obtained to determine how binding of the protein was affected
NOTE Both the absorption spectra and emission spectra are indicative that an equilibrium is reached with 42 μM Lysozyme and concentrations below 5 mM CaCl2. The protein concentration reaches at max absorbance at 500 μM CaCl2 and then decreases at 5 mM and 50 mM, respectively, indicating that lysozyme is precipitating out of the solution.
6. Solution Preparation
- Prepare new 0.6 g/L Lysozyme
- 0.0380 g of lysozyme dissolved in 50 mL H2O
- Prepare 5 mM Stock HCl solution
- Add 0.5 mL of 0.5 M HCL to 50 mL distilled H2O
- Dilutions were prepared from 5 mM HCl stock solution
- 75 μM, 100 μM, 250 μM, and 500 μM
- Dilutions were prepared from 5 mM HCl stock solution
- Add 0.5 mL of 0.5 M HCL to 50 mL distilled H2O
7. Prepare New Dialysis
- 42 μM Lysozyme (0.6 g/L) vs HCl
- Use 20,000 MWCO tubing
- Add 1 mL 0.6 g/L Lysozyme to 5 cells on one side of chamber
- Add 1 mL 75 μM, 100 μM, 250 μM, 500 μM, and 5 mM HCl to the opposite side of the chamber (one concentration per cell)
- Secure wells by screwing them to prevent any evaporation
- Place on low speed shaker for one week and prepare for analysis
