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

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==Entry title==
* Insert content here...


==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 [[User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2013/09/24 | see here]] didn't give the best results. The protocol for today is based off of [http://www.worthington-biochem.com/pm/assay.html 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
# Pepsin Digest
## Stock Solutions
### 2.5 w/v Hemoglobin in 0.06N HCl
### 1M perchloric acid
### 0.5mg/mL pepsin in 0.01N HCl
### pepsin-AuNP (at ratio that I need to decide on)
## 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)
### Make 1mL pepsin dilutions for the regular pepsin AND the pepsin-AuNPs in 0.01N HCl
#### 10μg/mL
#### 15μg/mL
#### 20μg/mL
### Blank (perform this for each pepsin or pepsin-AuNP dilution)
#### Place 2.5mL hemoglobin solution in a test tube in the 37C water bath
#### Add 5mL of perchloric acid
#### Add 0.5mL pepsin dilution
#### Remove from water bath after 5minutes
#### Filter
#### Measure the absorbance at 280 --- This will be your A<sub>280</sub>(Blank)
### Test (perform this for each pepsin or pepsin-AuNP dilution)
#### Place 2.5mL hemoglobin solution in a test tube in the 37C water bath
#### Add 0.5 mL pepsin dilution
#### Wait 10 minutes
#### Add 5mL of perchloric acid
#### Remove from water bath after 5minutes
#### Filter
#### Measure the absorbance at 280 --- This will be your A<sub>280</sub>(Test)
### Calculate the number of units/mg of active enzyme in each sample
#### Units/mg = [A<sub>280</sub>(Test) - A<sub>280</sub>(Blank)]*1000/[10minutes*mg enzyme in reaction mixture]</s>
# Protein-AuNP analysis
## Take a picture and note physical description of each ratio
## Place 1mL of each ratio in an 1.5mL eppendorf tube.
## 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.)
## Take a UV-Vis spectrum of each sample (Be considerate of other users here)
## After taking the spectrum, save the sample in a fresh eppy tube.
# Synthesis of HRP-AuNPs and Hemoglobin-AuNPs
## Into test tube that will contain a total of 5mL add
## Au solution such that the final concentration is 0.25mM
## For Hemoglobin make the following solutions to reflect an Au:Protein ratios
### 30
### 50
### 70
### 90
### 110
### 130
### 150
### 170
### 190
### 210
## For HRP make the following solutions to reflect an Au:Protein ratios
### 230
### 250
### 270
### 290
### 310
### 330
### 350
### 370
### 390
### 410
## Water up to 5mL
==Data==
* Add data and results here...
==Notes==


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Revision as of 09:25, 23 October 2013

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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]
  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


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