User:Benjamin Friedel/Notebook/CHEM 471/2015/10/14: Difference between revisions

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#After incubation of a sample and blank pair, they were centrifuged at 12,000rpm for 1 min.  
#After incubation of a sample and blank pair, they were centrifuged at 12,000rpm for 1 min.  
## Spun down the AuNP fiber sample and chymotrypsin blank at 12,000 RPM for 1 minute
## Spun down the AuNP fiber sample and chymotrypsin blank at 12,000 RPM for 1 minute
## Then the fluorescence assay reaction mix was prepared in a new 1.5ml eppendorf tube and contained the components and volumes shown below.Diluted bradford reagent consisted of a 1/4 dilution of the original bradford stock available in Dr. Harting's lab (not the bradford reagent from Dr. Saldanha's lab).
## Then the bradford assay reaction mix was prepared in a new plastic absorbance cuvettes and contained the components and volumes shown below.Diluted bradford reagent consisted of a 1/4 dilution of the original bradford stock available in Dr. Harting's lab (not the bradford reagent from Dr. Saldanha's lab).
### 750uL of the incubated blank or sample
### 750uL of the incubated blank or sample
### 1650uL of Tris Buffer
### 1650uL of Tris Buffer

Revision as of 11:58, 22 November 2015

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Objective

The objective for today is to measure the UV absorbance of lysozyme AuNP fibers over various incubation times with alpha chymotrypsin using a bradford assay.

Protocol

Protease Preparation

  1. 1mL stock solution of alpha-chymotrypsin was made by adding 1mL of 50mM phosphate buffer (pH=8) to dry protease for a final concentration of 41.016µM.
    1. Through M1V1= M2V2 the correct volume of protease needed for a final incubation tube concentration of 1uM and final tube volume of 1mL was determined
      1. V1= 2.44uL
  2. Each sample and blank tube for incubation requires a total volume of 1mL, thus 1mLtotal - 2.44uL protease = 997.6uL phosphate buffer.

Incubation Tube Preparation (Samples and Blanks)

  1. Fiber samples synthesized by Dr. Hartings were used for total of 14 (7 sample and 7 blank) and organized via the following alpha chymotrypsin incubation times.
      1. 10min, 15min, 30min, 45min, 1hr, 1.5hr, 2 hr
    2. First, AuNP fiber samples were centrifuged at 300 rpm for 10 minutes after which as much supernatant was drained as possible.
  2. Then, using volumes indicated above, alpha chymotrypsin and phosphate buffer were added to each sample and blank tube immediately before incubation start time.
    1. Sample and blank pairs were incubated at 37˚ C in a water bath for the noted incubation times above.


Bradford Assay Preparation

  1. After incubation of a sample and blank pair, they were centrifuged at 12,000rpm for 1 min.
    1. Spun down the AuNP fiber sample and chymotrypsin blank at 12,000 RPM for 1 minute
    2. Then the bradford assay reaction mix was prepared in a new plastic absorbance cuvettes and contained the components and volumes shown below.Diluted bradford reagent consisted of a 1/4 dilution of the original bradford stock available in Dr. Harting's lab (not the bradford reagent from Dr. Saldanha's lab).
      1. 750uL of the incubated blank or sample
      2. 1650uL of Tris Buffer
      3. 600uL of diluted Bradford reagent
      4. UV Absorbance was then immediately measured from 400-800nm
  2. Note that a spectra with the same parameters was taken for a sample with the same makeup as shown above, with the replacement of the incubated sample with Tris buffer for a bradford blank.




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