User:Javier Vinals Camallonga/Notebook/Javier Vinals notebook/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.
For this experiment, we run 5 samples of Pepsin and Pepsatin in the Uv-Vis after shaking. We took a sample every half an hour, and then we took one sample overnight.
"Figure 1. Corrected absorbance of Pepsin 2 nM"
"Figure 2. Corrected absorbance of pepsatin 2nM"
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