User:Benjamin Friedel/Notebook/CHEM 471/2015/10/14: Difference between revisions
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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''' | |||
# 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. | |||
## 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 | |||
### V1= 2.44uL | |||
# 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)''' | |||
#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. | |||
### 10min, 15min, 30min, 45min, 1hr, 1.5hr, 2 hr | |||
## First, AuNP fiber samples were centrifuged at 300 rpm for 10 minutes after which as much supernatant was drained as possible. | |||
# Then, using volumes indicated above, alpha chymotrypsin and phosphate buffer were added to each sample and blank tube immediately before incubation start time. | |||
## Sample and blank pairs were incubated at 37˚ C in a water bath for the noted incubation times above. | |||
'''Bradford Assay Preparation''' | |||
#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 | |||
## 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). | |||
### 750uL of the incubated blank or sample | |||
### 1650uL of Tris Buffer | |||
### 600uL of diluted Bradford reagent | |||
### UV Absorbance was then immediately measured from 400-800nm | |||
# 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. | |||
Revision as of 11:58, 22 November 2015
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ObjectiveThe objective for today is to measure the UV absorbance of lysozyme AuNP fibers over various incubation times with alpha chymotrypsin using a bradford assay. ProtocolProtease Preparation
Incubation Tube Preparation (Samples and Blanks)
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