User:Matthew R Skorski/Notebook/471 - Exp BioChem/2015/11/11: Difference between revisions
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To analyze the activity of proteinase k at 1 nM using fluorescence | To analyze the activity of proteinase k at 1 nM using fluorescence | ||
==Description== | |||
The guide for analyzing a protease with fluorescence can be found [[User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2015/10/06| here]] at Dr. Hartings notebook | |||
#Prepping AuNP Fiber Samples | |||
##7 AuNP fiber samples were spun at 300 RPM for 10 minutes | |||
##The supernatant was removed but the fibers were retained | |||
##Protinase K tube 11 was mixed with 1 mL of 1 mL of 50 mM pH 8 phosphate buffer | |||
###Protinase K concentration: (___)*(1mol/28,900g)*(1/0.001L)= 0.0000___ M Protinase K | |||
###Amount of Proteinase K solution needed for 1mL with 1μM concentration: M1*V1 = M2*V2 => (____ μM)*(V1) = (100 nM)*(1 mL) => V1 = 0.00__ mL | |||
###Amount of Buffer solution need to get to 1mL: (1mL total)-(0.____ mL Protinase K solution) = 0.____ mL buffer | |||
NEED TO GET DATA AS WELL AS DILUTION FROM SYDNEY | |||
#Incubating Samples | |||
##____ μL Protinase K and ____ μL buffer were mixed in the 7 fiber tubes as well as 7 blank eppendorf tubes | |||
##Eppendorf tubes were incubated on a shaker in a 37°C water bath for 25 minutes, 45 minutes, 75 minutes, 45 minutes, 105 minutes, 135 minutes, and 1440 minutes for one of the fiber tubes and one of the blanks. | |||
#Running Samples | |||
##Once the time for a sample was up the tube was centrifuged at 12000 rpm for 1 minute to collect the remaining fibers | |||
##From either the sample or blank, 20uL was taken and placed in a 600uL eppendorf tube | |||
###Added 140uL of Assay Buffer | |||
###Added 40uL of Assay Reagent | |||
##Taking Measurement | |||
###Excitation at 390 nm | |||
###Emission from 400 to 650 nm | |||
###Both slit widths set to 10 nm | |||
###Scan Speed of 200 | |||
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Revision as of 19:40, 24 November 2015
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ObjectiveTo analyze the activity of proteinase k at 1 nM using fluorescence DescriptionThe guide for analyzing a protease with fluorescence can be found here at Dr. Hartings notebook
NEED TO GET DATA AS WELL AS DILUTION FROM SYDNEY
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