User:Moira M. Esson/Notebook/CHEM-571/2013/09/24: Difference between revisions
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== | ==Objectives== | ||
* | #Observe the catalytic activity of pepsin and pepstatin. | ||
#Collect four samples each of pepsin and pepstatin for SDS-PAGE to be run tomorrow. | |||
#Run UV-vis to observe the effects of pepsin and pepstatin. | |||
<br> | |||
==General Protocol== | |||
The following protocol was derived from [http://pubs.acs.org/doi/abs/10.1021/la001164w this reference]. | |||
<br> | |||
#Combine 5mL hemoglobin with 8.333μL of 1.2E-6M pepsin to make a solution with a final concentration of 2nM. | |||
#Prepare a 5mL sample of hemoglobin with 83.3μL of 1.2mM pepstatin to make a solution with a final concentration of 20μM. | |||
#Incubate these solutions at 37°C | |||
#After 30 minutes, remove the two samples to prepare samples for SDS-PAGE and UV-vis | |||
##SDS-PAGE sample preparation | |||
##*Remove 10μL sample and dilute the sample to 1mL with 50mM Glycine-HCl buffer. | |||
##*Take 10μL of the diluted sample and add 10μL of SDS-PAGE buffer. | |||
##*Store prepared samples in the fridge | |||
###UV-vis sample preparation | |||
###*Remove 0.75mL of sample(either pepsin or pepstatin) and add 0.75mL perchloric acid | |||
#Repeat step 4 for two hours. | |||
Revision as of 12:33, 24 September 2013
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Objectives
General ProtocolThe following protocol was derived from this reference.
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