User:Eleni N. Kalivas/Notebook/CHEM-571/2013/09/24: Difference between revisions
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== | ==Protocol== | ||
The following protocol was taken from [User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2013/09/24 Matt Hartings] | |||
We will allow the pepsin cleavage of hemoglobin to proceed for two hours with aliquots taken and analyzed every 1/2 hour. We will also run parallel reactions with different concentrations of pepstatin (pepsin inhibitor) present. We will save our aliquots for analysis tomorrow using SDS-PAGE. | |||
# Combine 5 mL of the hemoglobin solution with the appropriate amount of pepsin (the final concentration should be 2 nM). | |||
## Do the same only with an appropriate amount of pepstatin added. (Each group will use a different concentration of pepstatin. We'll want 2 nM, 20 nM, 0.2uM, 2uM, and 20uM) | |||
# Incubate these solutions at 37C (in the incubator shaker) | |||
# After 30 minutes, remove two samples, as instructed below, for analysis today and tomorrow. | |||
## Prepare a sample for SDS-PAGE tomorrow | |||
### Remove 10uL of the reaction sample and dilute to 1mL with the Glycine-HCl buffer | |||
### Take 10uL of this diluted sample and mix with 10uL of the SDS-PAGE running buffer | |||
### Store all of your SDS-PAGE samples in the fridge overnight. | |||
## Prepare a sample for UV-Vis analysis today | |||
### Remove 0.75mL of the reaction sample and add 0.75mL of 1.7M perchloric acid to precipitate the remaining hemoglobin | |||
### After this sample sits for 1 hour, centrifuge for 15 minutes (organize centrifuge time with the other groups) in order to remove solid (uncleaved hemoglobin) from solution | |||
### Measure the absorption spectrum (specifically note 280nm) in order to determine the protein concentration in solution. Use the stock hemoglobin solution as your reference | |||
# Repeat Step 3 every 30 minutes for 2 hours. | |||
Revision as of 09:46, 24 September 2013
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ProtocolThe following protocol was taken from [User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2013/09/24 Matt Hartings] We will allow the pepsin cleavage of hemoglobin to proceed for two hours with aliquots taken and analyzed every 1/2 hour. We will also run parallel reactions with different concentrations of pepstatin (pepsin inhibitor) present. We will save our aliquots for analysis tomorrow using SDS-PAGE.
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