User:James C. Schwabacher/Notebook/CHEM-571/2014/01/29: Difference between revisions

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The objective for today is to begin the synthesis of BSA:L-Cysteine Nanocrystals
The objective for today is to begin the synthesis of BSA:L-Cysteine Nanocrystals


==Preparation of stock solutions==
 
 
==Protocol BSA:L-cysteine Nanocrystals==
# In test tube mix 180 uL of 5mM Zinc Nitrate and 20 uL of 5mM Manganese Acetate
# Add 200 uL of 40 mM Ammonium Acetate
# Add 200 uL of Protein:ligand in various ratios from 0% to 100% protein
# Adjust pH to 8.2 using roughly 250 uL of 10mM Ammonium Hydroxide
# Incubate at room temp for 1 hour
# Add 200 uL of 5mM Sodium Sulfide drop wise, then cover the test tubes
#A VWR Analog Vortex Mixer was used to vortex each test tube for 30 seconds
# Incubate for five-six days at 37 degrees Celsius
 
==Sample Preparation==
#Zinc Nitrate
## 0.0372g in 25mL --> 5mM
#Manganese Acetate
##Stock: 0.034g in 25mL --> 5.55mM
##Final: 9mL stock in 10mL --> 5mM
#Ammonium Acetate
##0.0321g in 10mL -->41.65mM
# BSA
# BSA
## .0064 g of BSA in 10mL of water --> 9.69 μM
## .0064 g of BSA in 10mL of water --> 9.69 μM
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## 6.76 μL of 28-30% Ammonium Hydroxide in 10 mL
## 6.76 μL of 28-30% Ammonium Hydroxide in 10 mL
#Sodium Sulfide
#Sodium Sulfide
##Stock: 0.0495g in 25 mL --> 8.24mM
##Final: 6.06mL of stock in 10mL --> 5mM


==Protocol BSA:L-cysteine Nanocrystals==
{| {{table}}
# In test tube mix 180 uL of 5mM Zinc Nitrate and 20 uL of 5mM Manganese Acetate
| align="center" style="background:#f0f0f0;"|'''% BSA'''
# Add 200 uL of 40 mM Ammonium Acetate  
| align="center" style="background:#f0f0f0;"|'''5 mM Zinc Nitrate (uL)'''
# Add 200 uL of Protein:ligand in various ratios from 0% to 100% protein
| align="center" style="background:#f0f0f0;"|'''5mM Manganese Acetate (uL)'''
# Adjust pH to 8.2 using roughly <del>250 uL</del> 1 mL of 10mM Ammonium Hydroxide, <del>and bring volume of water to 2.3 mL</del>
| align="center" style="background:#f0f0f0;"|'''41.65 mM Ammonium Acetate (uL)'''
# Incubate at room temp for 1 hour
| align="center" style="background:#f0f0f0;"|'''9.69mM BSA (uL)'''
# Add 200 uL of 5mM Sodium Sulfide drop wise while vortexing
| align="center" style="background:#f0f0f0;"|'''10mM L-Cysteine (uL)'''
# Incubate for five days at 37 degrees Celsius
| align="center" style="background:#f0f0f0;"|'''10mM Ammonium Hydroxide (uL)'''
| align="center" style="background:#f0f0f0;"|'''5 mM Sodium Sulfide (uL)'''
| align="center" style="background:#f0f0f0;"|'''Total Volume (mL)'''
|-
| 0||180||20||200||0||200||1150||200||1.95
|-
| 10||180||20||200||20||180||1000||200||1.8
|-
| 20||180||20||200||40||160||1000||200||1.8
|-
| 30||180||20||200||60||140||1000||200||1.8
|-
| 40||180||20||200||80||120||1000||200||1.8
|-
| 50||180||20||200||100||100||1000||200||1.8
|-
| 60||180||20||200||120||80||1000||200||1.8
|-
| 70||180||20||200||140||60||1000||200||1.8
|-
| 80||180||20||200||160||40||1000||200||1.8
|-
| 90||180||20||200||180||20||1450||200||2.25
|-
| 100||180||20||200||200||0||1100||200||1.9
|}


==Table of BSA:L-Cysteine ratios==  
==Table of BSA:L-Cysteine ratios==  

Revision as of 16:44, 29 January 2014

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objective

The objective for today is to begin the synthesis of BSA:L-Cysteine Nanocrystals


Protocol BSA:L-cysteine Nanocrystals

  1. In test tube mix 180 uL of 5mM Zinc Nitrate and 20 uL of 5mM Manganese Acetate
  2. Add 200 uL of 40 mM Ammonium Acetate
  3. Add 200 uL of Protein:ligand in various ratios from 0% to 100% protein
  4. Adjust pH to 8.2 using roughly 250 uL of 10mM Ammonium Hydroxide
  5. Incubate at room temp for 1 hour
  6. Add 200 uL of 5mM Sodium Sulfide drop wise, then cover the test tubes
  7. A VWR Analog Vortex Mixer was used to vortex each test tube for 30 seconds
  8. Incubate for five-six days at 37 degrees Celsius

Sample Preparation

  1. Zinc Nitrate
    1. 0.0372g in 25mL --> 5mM
  2. Manganese Acetate
    1. Stock: 0.034g in 25mL --> 5.55mM
    2. Final: 9mL stock in 10mL --> 5mM
  3. Ammonium Acetate
    1. 0.0321g in 10mL -->41.65mM
  4. BSA
    1. .0064 g of BSA in 10mL of water --> 9.69 μM
  5. L-Cysteine
    1. .0259 g in 25 mL water --> 8.55 mM
    2. 11.7 uL in 10 mL water --> 10μM
  6. Ammonium Hydroxide
    1. 6.76 μL of 28-30% Ammonium Hydroxide in 10 mL
  7. Sodium Sulfide
    1. Stock: 0.0495g in 25 mL --> 8.24mM
    2. Final: 6.06mL of stock in 10mL --> 5mM
% BSA 5 mM Zinc Nitrate (uL) 5mM Manganese Acetate (uL) 41.65 mM Ammonium Acetate (uL) 9.69mM BSA (uL) 10mM L-Cysteine (uL) 10mM Ammonium Hydroxide (uL) 5 mM Sodium Sulfide (uL) Total Volume (mL)
0 180 20 200 0 200 1150 200 1.95
10 180 20 200 20 180 1000 200 1.8
20 180 20 200 40 160 1000 200 1.8
30 180 20 200 60 140 1000 200 1.8
40 180 20 200 80 120 1000 200 1.8
50 180 20 200 100 100 1000 200 1.8
60 180 20 200 120 80 1000 200 1.8
70 180 20 200 140 60 1000 200 1.8
80 180 20 200 160 40 1000 200 1.8
90 180 20 200 180 20 1450 200 2.25
100 180 20 200 200 0 1100 200 1.9

Table of BSA:L-Cysteine ratios

mol of protein 2.00E-09 1.80E-09 1.60E-09 1.40E-09 1.20E-09 1.00E-09 8.00E-10 6.00E-10 4.00E-10 2.00E-10 0
percentage of protein 100 90 80 70 60 50 40 30 20 10 0
percentage of ligand 0 10 20 30 40 50 60 70 80 90 100
mol of ligand 0 2.00E-10 4.00E-10 6.00E-10 8.00E-10 1.00E-09 1.20E-09 1.40E-09 1.60E-09 1.80E-09 2.00E-09

Notes

This area is for any observations or conclusions that you would like to note.


Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.




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