User:Khyra A. Neal/Notebook/Chem 571/2014/10/29: Difference between revisions
From OpenWetWare
(Autocreate 2014/10/29 Entry for User:Khyra_A._Neal/Notebook/Chem_571) |
(fix raw html notebook nav) |
||
(5 intermediate revisions by one other user not shown) | |||
Line 2: | Line 2: | ||
|- | |- | ||
|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}}}}]]}} | ||
|- | |- | ||
| colspan="2"| | | colspan="2"| | ||
<!-- ##### 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. ##### --> | ||
== | ==October 29, 2014== | ||
===Tasklist=== | |||
1. Extract Lysozyme and CaCl<sub>2</sub> solutions | |||
* Solutions were prepared on [http://openwetware.org/wiki/User:Khyra_A._Neal/Notebook/Chem_571/2014/10/28 October 28, 2014] | |||
* Extracted with a pasteur pipette and transferred to 30 mL extraction vials | |||
2. Bradford Analysis | |||
* Extracted Lysozyme solutions that were dialyzed against CaCl<sub>2</sub> | |||
* 200 μL Bradford Reagent (diluted 1:3) | |||
* 50 μL Sample Solution | |||
* 750 μL 50 mM Tris/ 50 mM NaCl Buffer | |||
* Run UV-Vis between 400 nm and 800 nm | |||
[[Image:Bradford_of_Lys_vs_CaCl2.jpg]] | |||
3. | |||
<u>Ca<sup>2+</sup> ISE</u> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''CaCl<sub>2</sub> concentration''' | |||
| align="center" style="background:#f0f0f0;"|'''mV ''' | |||
|- | |||
| 5μM CaCl<sub>2</sub> || 11.8 | |||
|- | |||
| Lys (1 ) || 4.6 | |||
|- | |||
| 50μM CaCl<sub>2</sub> || 4.2 | |||
|- | |||
| Lys (2) || 0.1 | |||
|- | |||
| 500μM CaCl<sub>2</sub> || 27.4 | |||
|- | |||
| Lys (3) || 24.4 | |||
|- | |||
| 5mM CaCl<sub>2</sub> || 52.1 | |||
|- | |||
| Lys (4) || 51.3 | |||
|- | |||
| 50mM CaCl<sub>2</sub>|| 77.3 | |||
|- | |||
| Lys (5) || 77.0 | |||
|- | |||
|} | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | ||
|} | |} | ||
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 | |||
[[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
Project name | Main project page Previous entry Next entry | ||||||||||||||||||||||
October 29, 2014Tasklist1. Extract Lysozyme and CaCl2 solutions
2. Bradford Analysis
3. Ca2+ ISE
|
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