User:Zachary I. Mendel/Notebook/Zacks Notebook/2013/10/23

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==Notes==
==Notes==
 +
 +
Table 1. Stock solutions used in the preparation of Protein-AuNP solutions
 +
<br>
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Concentration HRP Day 2(M)'''
 +
| align="center" style="background:#f0f0f0;"|'''Concentration Gold Stock (M)'''
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| align="center" style="background:#f0f0f0;"|'''Myoglobin (M)'''
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|-
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| 0.0000015||0.0025||0.0000165
 +
|}
 +
<br>
 +
Table 2. Preparation of HRP-AuNP solutions
 +
<br>
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Ratio'''
 +
| align="center" style="background:#f0f0f0;"|'''Amount of gold added (mL)'''
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| align="center" style="background:#f0f0f0;"|'''Concentration HRP (M)'''
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| align="center" style="background:#f0f0f0;"|'''Volume HRP (mL)'''
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| align="center" style="background:#f0f0f0;"|'''Volume H<sub>2</sub>O added (mL)'''
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|-
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| 230||0.5||1.08696E-06||3.623188406||0.876811594
 +
|-
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| 250||0.5||0.000001||3.333333333||1.166666667
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|-
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| 270||0.5||9.25926E-07||3.086419753||1.413580247
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|-
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| 290||0.5||8.62069E-07||2.873563218||1.626436782
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|-
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| 310||0.5||8.06452E-07||2.688172043||1.811827957
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|-
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| 330||0.5||7.57576E-07||2.525252525||1.974747475
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|-
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| 350||0.5||7.14286E-07||2.380952381||2.119047619
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|-
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| 370||0.5||6.75676E-07||2.252252252||2.247747748
 +
|-
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| 390||0.5||6.41026E-07||2.136752137||2.363247863
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|-
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| 410||0.5||6.09756E-07||2.032520325||2.467479675
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|}
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<br>
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Table 3. Preparation of Myoglobin-AuNPs
 +
<br>
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Ratio'''
 +
| align="center" style="background:#f0f0f0;"|'''Amount of gold added (mL)'''
 +
| align="center" style="background:#f0f0f0;"|'''Concentration Myoglobin (M)'''
 +
| align="center" style="background:#f0f0f0;"|'''Volume of myogloblin(mL)'''
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| align="center" style="background:#f0f0f0;"|'''Volume Water Myoglobin(mL)'''
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|-
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| 30||0.5||8.33333E-06||2.525252525||1.974747475
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|-
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| 50||0.5||0.000005||1.515151515||2.984848485
 +
|-
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| 70||0.5||3.57143E-06||1.082251082||3.417748918
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|-
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| 90||0.5||2.77778E-06||0.841750842||3.658249158
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|-
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| 110||0.5||2.27273E-06||0.688705234||3.811294766
 +
|-
 +
| 130||0.5||1.92308E-06||0.582750583||3.917249417
 +
|-
 +
| 150||0.5||1.66667E-06||0.505050505||3.994949495
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|-
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| 170||0.5||1.47059E-06||0.445632799||4.054367201
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|-
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| 190||0.5||1.31579E-06||0.398724083||4.101275917
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|-
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| 210||1.19048E-06||0.360750361||4.139249639
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|}
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<br>
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 +
 +
Figure 1: Corrected Absorbance Spectra for Pepsin AuNPs
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[[Image:Pepsin Nano.png]]
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Revision as of 10:02, 28 October 2013

Project name Main project page
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Objective

To measure pepsin activity of 1) pepsin in solution and 2) pepsin-AuNPs in solution. We're going to try a slightly different protocol for this since the previous one see here didn't give the best results. The protocol for today is based off of this.

We'll also be taking spectra of the pepsin-AuNP samples you made yesterday.

Description

Make a 37C water bath that can hold at least 3 test tubes (same size as you used yesterday) at a time --- START THIS FIRST

  1. Pepsin Digest
    1. Stock Solutions
      1. 2.5 w/v Hemoglobin in 0.06N HCl
      2. 1M perchloric acid
      3. 0.5mg/mL pepsin in 0.01N HCl
      4. pepsin-AuNP (at ratio that I need to decide on)
    2. Directions (You can decide how you want to split your effort here. I'd suggest doing one set, i.e. pepsin blank, pepsin trial, pepsin-AuNP blank, or pepsin-AuNP trial, at a time)
      1. Make 1mL pepsin dilutions for the regular pepsin AND the pepsin-AuNPs in 0.01N HCl
        1. 10μg/mL
        2. 15μg/mL
        3. 20μg/mL
      2. Blank (perform this for each pepsin or pepsin-AuNP dilution)
        1. Place 2.5mL hemoglobin solution in a test tube in the 37C water bath
        2. Add 5mL of perchloric acid
        3. Add 0.5mL pepsin dilution
        4. Remove from water bath after 5minutes
        5. Filter
        6. Measure the absorbance at 280 --- This will be your A280(Blank)
      3. Test (perform this for each pepsin or pepsin-AuNP dilution)
        1. Place 2.5mL hemoglobin solution in a test tube in the 37C water bath
        2. Add 0.5 mL pepsin dilution
        3. Wait 10 minutes
        4. Add 5mL of perchloric acid
        5. Remove from water bath after 5minutes
        6. Filter
        7. Measure the absorbance at 280 --- This will be your A280(Test)
      4. Calculate the number of units/mg of active enzyme in each sample
        1. Units/mg = [A280(Test) - A280(Blank)]*1000/[10minutes*mg enzyme in reaction mixture]</s>
  2. Protein-AuNP analysis
    1. Take a picture and note physical description of each ratio
    2. Place 1mL of each ratio in an 1.5mL eppendorf tube.
    3. Centrifuge for 30 minutes (Be considerate of other uses here. Act quickly, and get as many tubes in the centrifuge as you can at once.)
    4. Take a UV-Vis spectrum of each sample (Be considerate of other users here)
    5. After taking the spectrum, save the sample in a fresh eppy tube.
  3. Synthesis of HRP-AuNPs and Hemoglobin-AuNPs
    1. Into test tube that will contain a total of 5mL add
    2. Au solution such that the final concentration is 0.25mM
    3. For Hemoglobin make the following solutions to reflect an Au:Protein ratios
      1. 30
      2. 50
      3. 70
      4. 90
      5. 110
      6. 130
      7. 150
      8. 170
      9. 190
      10. 210
    4. For HRP make the following solutions to reflect an Au:Protein ratios
      1. 230
      2. 250
      3. 270
      4. 290
      5. 310
      6. 330
      7. 350
      8. 370
      9. 390
      10. 410
    5. Water up to 5mL

Data

  • Add data and results here...

Notes

Table 1. Stock solutions used in the preparation of Protein-AuNP solutions

Concentration HRP Day 2(M) Concentration Gold Stock (M) Myoglobin (M)
0.00000150.00250.0000165


Table 2. Preparation of HRP-AuNP solutions

Ratio Amount of gold added (mL) Concentration HRP (M) Volume HRP (mL) Volume H2O added (mL)
2300.51.08696E-063.6231884060.876811594
2500.50.0000013.3333333331.166666667
2700.59.25926E-073.0864197531.413580247
2900.58.62069E-072.8735632181.626436782
3100.58.06452E-072.6881720431.811827957
3300.57.57576E-072.5252525251.974747475
3500.57.14286E-072.3809523812.119047619
3700.56.75676E-072.2522522522.247747748
3900.56.41026E-072.1367521372.363247863
4100.56.09756E-072.0325203252.467479675


Table 3. Preparation of Myoglobin-AuNPs

Ratio Amount of gold added (mL) Concentration Myoglobin (M) Volume of myogloblin(mL) Volume Water Myoglobin(mL)
300.58.33333E-062.5252525251.974747475
500.50.0000051.5151515152.984848485
700.53.57143E-061.0822510823.417748918
900.52.77778E-060.8417508423.658249158
1100.52.27273E-060.6887052343.811294766
1300.51.92308E-060.5827505833.917249417
1500.51.66667E-060.5050505053.994949495
1700.51.47059E-060.4456327994.054367201
1900.51.31579E-060.3987240834.101275917
2101.19048E-060.3607503614.139249639



Figure 1: Corrected Absorbance Spectra for Pepsin AuNPs Image:Pepsin Nano.png


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