User:Daniel-Mario Larco/Notebook/AU Biodesign Lab - 09/03/2013/2013/09/24
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To observe the catalytic activity of pepsin (in cleaving peptide bonds in hemoglobin) in the presence and absence of pepstatin. This data will be compared with data we take in a later lab on pepsin-AuNPs.
The experimental details (pepsin cleavage of hemoglobin) are similar to this reference.
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.
Making the solutions
Glycine-HCl Buffer pH 3
50 mL of 5 mg/mL Hemoglobin
Pepstatin is not very soluble in water. Solubilize in methanol as per wikipedia.
4.0mg pepstatin in 5mL methanol ---> 1.2mM pepstatin
SDS-PAGE Sample Buffer (No DTT)
4.2mg pepsin in 10mL (MW of pepsin is 34620 according to sigma) ---> 1.2uM
The class ran out of some solutions and Karlena made new:
250.1 mg hemoglobin in 50mL water
Glycine HCl buffer
0.37910g glycine in 100mL water and adjust the pH to 3 ---> 50.5mM Glycine-HCl buffer pH 3
We ran the UV-Vis of 5 samples containing hemoglobin and pepsin and 5 samples containing hemoglobin and pepsatin. The first 4 samples of each we made at 30 minute intervals starting from when the pepsin or pepsatin was added to the hemoglobin. The 5th samples, were taken the following day to see if any more changes had occurred overnight.
Here are the UV-Vis spectra for both sets of samples.