User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/14

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(Procedure)
Current revision (12:57, 21 November 2012) (view source)
(Procedure)
 

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Experimental Biological Chemistry I Main project page
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Purpose

  • Perform dialysis on purified ADA solution to decrease the concentration of salt in ADA solutions.
  • Make Au/ADA and Au/HRP samples with ratio between 60 and 150 in 10 increments

Procedure

  • Dialysis of ADA solution was performed in the following way:
    • 4000mL of distilled water was placed in a large 5000mL dialysis beaker.
    • For ADA solution purified in 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 two separate protein sample purified on 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.
    • 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.
    • 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:
  60 - 70 - 80 - 90 - 100 - 110 - 120 - 130 - 140 - 150
  • The preparation for Au/ADA solutions were as follows:
    • ADA used in this set of samples had a concentration of 58.37uM and was made from 10/03/2012.
    • The HAuCl4 stock solutions was made on 09/05/2012 and has a concentration of 10.50mM.
    • The water used was distilled water at 16 mega ohms.
    • ADA and HAuCl4 were added first, then water was added. All samples were wrapped with aluminum foil around to minimize exposure to light, and placed in 85°C incubator for 4 hours. After 4 hours of incubation, the samples were left in room temperature.
    • Below table listed the amount of ADA, HAuCl4, and water added for samples for all ratios:
Au/ADA ratio ADA added[uL] HAuCl4[uL] Water[uL]
60137.145.717817.2
70137.153.37809.6
80137.160.97802.0
90137.168.67794.4
100137.176.27786.8
110137.183.87779.1
120137.191.47771.5
130137.199.07763.9
140137.1106.77756.3
150137.1114.37748.7
  • The Au/HRP solutions were also made in the same ratio as Au/ADA, ranging from 60 to 120 in increments of 10.
    • Sample with ratio 130, 140 and 150 were made with newly made HRP stock solution, details of making the HRP stock solution and determination of the concentration were described later on.
    • The HAuCl4 stock solution was prepared from 09/05/2012 with a concentration of 10.50mM.
    • The HRP stock solution was made on 09/05/2012 and by mixing 0.0057mg of HRP with 5mL of autoclaved water.
    • Water used to make Au/HRP solution were deionized through 16 mega ohms filter.
    • HAuCl4 and HRP were placed in small test tubes first, then water were added. The samples were wrapped with aluminum foil and placed in incubator under the same protocol as Au/ADA samples.
    • The volumes of HAuCl4, HRP, and water were listed in table below:
Au/HRP ratio HRP added[uL] HAuCl4[uL] Water[uL]
60280.745.717673.6
70280.753.37666.0
80280.760.97658.4
90280.768.67650.7
100280.776.27643.1
110280.783.87635.5
120280.791.47627.9
  • For Au/HRP samples with the following ratios were made with newly made HRP stock solution:
   130 - 140 - 150
  • HRP stock solution were made by mixing 0.0057g of HRP with 5mL of distilled water.
    • However, after mixing, it was suspected that the balance for weighting out the HRP were inaccurate. As a result, UV-vis spectrometer was used. A spectrum from 600nm to 200nm were run with 200uL of newly made HRP sample running against 200uL of water in 10.00mm quartz cuvette.
    • At a wavelength of 402nm, the absorbance of HRP sample was 2.100.
    • With given molar absorptivity at 402nm (102 M*cm-1), the concentration of HRP were calculated in the following way:
  Absorbance = Molar absorptivity * Path length * Concentration
  2.100 = 102 M*cm-1 * 1cm * Concentration
  Concentration of HRP sample = 20.58uM
  • With the newly calculated concentration, the volume for HAuCl4 and HRP in sample Au/HRP ratio 130, 140, and 150 were recalculated, and volume of for each samples were shown below:
Au/HRP ratio HRP added[uL] HAuCl4[uL] Water[uL]
130383.799.07517.3
140383.7106.77509.7
150383.7114.37502.0
  • 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 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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