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 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
- 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.
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