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== | ==October 15, 2014== | ||
* | |||
===Tasklist=== | |||
==Electrophoresis== | |||
* Warm solutions for 5 minutes at 40°C that were prepared on [http://openwetware.org/wiki/User:Khyra_A._Neal/Notebook/Chem_571/2014/10/14 October 14, 2014] | |||
* Load samples to premade gel | |||
** Well 1: Precision Plus Protein All Blue Standards | |||
** Well 3: 0.12 g/L lysozyme Unk A | |||
** Well 4: 30:1 Lysozyme colloid | |||
** Well 5: 0.12 g/L Lysozyme | |||
** Well 6: 0.6 g/L Lysozyme | |||
** Wells 8-12 contain solutions prepared by Dr. Fox | |||
* Run for 40 minutes at 200V | |||
* Develop/Stain your gel | |||
** Place gel in Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes | |||
** Place gel in Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour | |||
** Place gel in Destain Solution (10% acetic acid, 90% water) for 15 minutes | |||
*** Repeat this step with fresh destain solution 2 more times | |||
The original protocol for electrophoresis was written by [http://openwetware.org/wiki/User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2013/09/25 Dr. Hartings] | |||
==Bradford Analysis== | |||
Bradford Analysis was conducted on the dialyzed 30:1 colloid against CaCl<sub>2</sub> solutions | |||
* Remove 20 μL of solution from each chamber (10 in all) and run Bradford analysis | |||
* Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl | |||
* Dilute to 1 mL by adding 780 μL stock 50 mM Tris/NaCl | |||
* Measure UV-Vis from 400 nm - 800 nm using polystyrene cuvettes. | |||
* Run blank of Tris/NaCl buffer | |||
* Run UV-Vis of undialyzed 30:1 colloid solution with Bradford reagent | |||
[[Image:Bradford_of_Colloid_in_CaCl2.jpg]] | |||
'''NOTE''' The observed dialysis solution of 50 mM CaCl<sub>2</sub> was colorless and the colloid precipitated out. Free Lysozyme is detected at 50 mM CaCl<sub>2</sub> because the colloid precipitated out of solution. | |||
==UV-Vis and Fluorescence of Dialyzed Colloid Solutions== | |||
Fluorescence | |||
* Dilute Lysozyme sample 100x for each well. | |||
* Transfer 100 μL to a small volume fluorescence cuvette | |||
* Run fluorescence from 300 nm -550 nm and excitation at 280 nm | |||
* Clean the cuvette after each use with several SDS, HCl, HPLC, & methanol washes | |||
UV-Vis of Colloid Solutions | |||
* Transfer 100 μL of dialyzed solution to small volume glass cuvette | |||
* Run Uv-Vis on protein containing solutions | |||
Run from 200 nm -400 nm | |||
==Ca<sup>2+</sup> ISE== | |||
ISE for protein containing Ca<sup>2+</sup> ions | |||
<u>Ca2+ ISE</u> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''CaCl<sub>2</sub> concentration''' | |||
| align="center" style="background:#f0f0f0;"|'''mV''' | |||
|- | |||
| 5μM || 31.8 | |||
|- | |||
| 50μM || 40.6 | |||
|- | |||
| 500μM || 40.1 | |||
|- | |||
| 5mM || 56.6 | |||
|- | |||
| 50mM || 78.5 | |||
|- | |||
|} | |||
===Determination of Dialyzed KI Samples extracted on [http://openwetware.org/wiki/User:Khyra_A._Neal/Notebook/Chem_571/2014/10/08 October 14, 2014]=== | |||
==Determination of KI Concentration by Titration== | |||
* Standardization: | |||
** 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''' | |||
'''NOTE''' The final concentration of NH<sub>4</sub>SCN was determined by titration standardization on [http://openwetware.org/wiki/User:Khyra_A._Neal/Notebook/Chem_571/2014/10/08 October 8, 2014] | |||
'''Titrations were performed as follows''' | |||
* 9.68 mM NH<sub>4</sub> is being 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 | |||
*** [KI] = 7.6 mM | |||
** Lysozyme against 5 mM KI | |||
*** 14.2 mL NH<sub>4</sub>SCN used | |||
*** [KI] = 25 mM | |||
** Lysozyme against 10 mM KI | |||
*** 14.0 mL NH<sub>4</sub>SCN used | |||
*** [KI] = 29 mM | |||
** Lysozyme against 25 mM KI | |||
*** 13.7 mL NH<sub>4</sub>SCN used | |||
*** [KI] = 34.8 mM | |||
** Lysozyme against 50 mM KI | |||
*** 12.5 mL NH<sub>4</sub>SCN used | |||
*** [KI] = 58 mM | |||
** 2 mM KI against Lysozyme | |||
*** 13.6 mL NH<sub>4</sub>SCN used | |||
*** [KI] = 36.7 mM | |||
** 5 mM KI against Lysozyme | |||
*** 13.4 mL NH<sub>4</sub>SCN used | |||
*** [KI] = 40.6 mM | |||
** 10 mM KI against Lysozyme | |||
*** 13.2 mL NH<sub>4</sub>SCN used | |||
*** [KI] = 44.4 mM | |||
** 25 mM KI against Lysozyme | |||
***12.6 mL NH<sub>4</sub>SCN used | |||
*** [KI] = 56.1 mM | |||
** 50 mM KI against Lysozyme | |||
***11.9 mL NH<sub>4</sub>SCN used | |||
*** [KI] = 69.6 mM | |||
'''NOTE''' Our values are indicative of experimental error. We found that our initial dilutions of KI (2-25 mM) were not prepared at the indicated concentrations. All diluted KI solutions were discarded and new solutions were prepared using fresh 50 mM KI on October 21, 2014. | |||
Latest revision as of 00:27, 27 September 2017
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October 15, 2014TasklistElectrophoresis
The original protocol for electrophoresis was written by Dr. Hartings Bradford AnalysisBradford Analysis was conducted on the dialyzed 30:1 colloid against CaCl2 solutions
UV-Vis and Fluorescence of Dialyzed Colloid SolutionsFluorescence
UV-Vis of Colloid Solutions
Run from 200 nm -400 nm Ca2+ ISEISE for protein containing Ca2+ ions Ca2+ ISE
Determination of Dialyzed KI Samples extracted on October 14, 2014Determination of KI Concentration by Titration
NOTE The final concentration of NH4SCN was determined by titration standardization on October 8, 2014
NOTE Our values are indicative of experimental error. We found that our initial dilutions of KI (2-25 mM) were not prepared at the indicated concentrations. All diluted KI solutions were discarded and new solutions were prepared using fresh 50 mM KI on October 21, 2014.
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