User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/14: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Experimental Biological Chemistry I</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Experimental Biological Chemistry I</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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** 4000mL of distilled water was placed in a large 5000mL dialysis beaker. | ** 4000mL of distilled water was placed in a large 5000mL dialysis beaker. | ||
** For ADA solution purified in [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/03|10/03/2012]], about 10cm of SnakeSkin Dialysis Tubing from Thermo Scientific with a diameter 22mm was used. The bottom of dialysis tubing was folded twice over and clipped with a dialysis clip. ADA protein in Binding buffer (at pH 7.5, with 20mM Tris, 0.5M NaCl, and 30mM Imidazole) was transferred from 15mL falcon tube into the dialysis tube. The air bubbles were excluded from dialysis tube. Top of dialysis tube was folded twice and sealed with another dialysis clip. | ** For ADA solution purified in [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/03|10/03/2012]], about 10cm of SnakeSkin Dialysis Tubing from Thermo Scientific with a diameter 22mm was used. The bottom of dialysis tubing was folded twice over and clipped with a dialysis clip. ADA protein in Binding buffer (at pH 7.5, with 20mM Tris, 0.5M NaCl, and 30mM Imidazole) was transferred from 15mL falcon tube into the dialysis tube. The air bubbles were excluded from dialysis tube. Top of dialysis tube was folded twice and sealed with another dialysis clip. | ||
** The same process was performed for the protein purified on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/06|11/06/2012]]. About 10cm of dialysis tubing was used for ADA elution fraction 1 and 3, and about 5cm of dialysis tubing was used for ADA elution fraction 2. | ** The same process was performed for the two separate protein sample purified on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/06|11/06/2012]], named Fraction 1 & 3, and fraction 2. About 10cm of dialysis tubing was used for ADA elution fraction 1 and 3, and about 5cm of dialysis tubing was used for ADA elution fraction 2. | ||
** One Slide-A-Lyzer Buoys Dialysis Cassette was attached with groove of buoy on edge of one of the dialysis clips to ensure the ADA solution in dialysis tube float in water. | ** One Slide-A-Lyzer Buoys Dialysis Cassette was attached with groove of buoy on edge of one of the dialysis clips to ensure the ADA solution in dialysis tube float in water. | ||
** All three dialysis tubes were placed in water in dialysis beaker with stir bar. | ** All three dialysis tubes were placed in water in dialysis beaker with stir bar. | ||
** Dialysis beaker was covered with aluminum foil to prevent loss of water in the process, and the beaker was placed on a stir plate in -40°C for 12 days. | ** Dialysis beaker was covered with aluminum foil to prevent loss of water in the process, and the beaker was placed on a stir plate in -40°C for 12 days. | ||
** Distilled water in dialysis beaker was changed after two days, and dialysis tubings were soaked in new distilled water for 10 days. | |||
* The Au/ADA solutions were made to ensure the ratio of fibers formed. The ratios of Au/ADA solutions were as follows: | * The Au/ADA solutions were made to ensure the ratio of fibers formed. The ratios of Au/ADA solutions were as follows: | ||
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Concentration of HRP sample = 20.58uM | Concentration of HRP sample = 20.58uM | ||
* With the newly calculated concentration, the volume for HAuCl<sub>4</sub> and HRP in sample Au/HRP ratio 130, 140, and 150 were recalculated, and volume of for each samples were shown below: | * With the newly calculated concentration, the volume for HAuCl<sub>4</sub> and HRP in sample Au/HRP ratio 130, 140, and 150 were recalculated, and volume of for each samples were shown below: | ||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Au/HRP ratio''' | |||
| align="center" style="background:#f0f0f0;"|'''HRP added[uL]''' | |||
| align="center" style="background:#f0f0f0;"|'''HAuCl4[uL]''' | |||
| align="center" style="background:#f0f0f0;"|'''Water[uL]''' | |||
|- | |||
| 130||383.7||99.0||7517.3 | |||
|- | |||
| 140||383.7||106.7||7509.7 | |||
|- | |||
| 150||383.7||114.3||7502.0 | |||
|} | |||
* Above samples were also placed in incubator with same protocol as that of Au/ADA and Au/HRP samples. | |||
==Notes== | |||
* After two hours of dialysis, 1mL of ADA sample made from [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/03|10/03/2012]] was taken out with a pipette and transferred into a 1.5mL microcentrifuge tube. The ADA sample with concentration of 58.37uM was kept separate from the dialysis process to run ADA enzyme kinematics. | |||
* Due to time concerns, ADA enzyme kinematics were not run, and is scheduled to run next week. | |||
* The ADA sample was stored in freezer to prevent enzyme degradation for prolonged period of time. | |||
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Latest revision as of 22:15, 26 September 2017
Experimental Biological Chemistry I | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Purpose
Procedure
60 - 70 - 80 - 90 - 100 - 110 - 120 - 130 - 140 - 150
130 - 140 - 150
Absorbance = Molar absorptivity * Path length * Concentration 2.100 = 102 M*cm-1 * 1cm * Concentration Concentration of HRP sample = 20.58uM
Notes
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