User:Andrew W Long/Notebook/CHEM-671 Research/2015/10/06

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Proteinase-K 1uM Bradford

The instructions for the day can be found here

Procedural modifications: fibers previously formed firm pellets when spun down at 1500rpm, so from this day onward fibers were spun down at 300 rpm for 10min.

Previously synthesized fiber samples were prepared in the following way. First, 7 fiber samples were centrifuged, and the supernatant was removed. Stock 4 of proteinase-K was suspended with 1mL of Tris buffer (50mM) with CaCl (10mM) at pH 8. The concentration of proteinase-K of the resulting solution was then determined to be 0.00039946. This was calculated using 0.0014g * (1mol / 28,900g) * (1000mL / 1L). We want a 1mL final sample with a 1uM concentration. The amount of the proteinase-K solution added to the reaction tubes was determined to be 23.35mL. This was calculated using M1V1=M2V2. 39.446uM * V1 = 1uM * 1mL. The amount of buffer required to make the solution 1mL was determined to be 0.975. This was calculated using by doing 1mL total - 23.35mL proteinase-K = XmL buffer.

Next, the 7 samples were incubated in a shaking hot water bath, as well as the 7 blanks. Once each sample had incubated for it's allotted time, it was pulled out of the bath, and centrifuged at 300rpm for 1min. 750mL was placed in a plastic cuvette, with 600mL dilute Bradford stock solution, and 1650uL buffer. The time periods were 10min, 15min, 30min, 45min, 1hr, 1.5hr, and 2hr. The samples were scanned from 400-800nm using the UV-Vis. The results from the experiment are shown below:

AMS 1uM ProteinaseK Fibers Bradford 6oct2015.png

Absorbance at 600nm 1uM Bradford AMS 6102015.png