User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2013/09/24: Difference between revisions
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==Data== | ==Data== | ||
Making the solutions | |||
<u>Glycine-HCl Buffer pH 3</u> | |||
# 0.7556g glycine in 200mL water adjust the pH with HCl ---> 50mM Gly-HCl | |||
<u>50 mL of 5 mg/mL Hemoglobin</u> | |||
# 0.2549g hemoglobin in 50mL of Gly-HCl buffer pH 3 ---> 51.0 mg/mL | |||
<u>Pepstatin</u> | |||
Pepstatin is not very soluble in water. Solubilize in methanol as per [http://en.wikipedia.org/wiki/Pepstatin wikipedia]. | |||
4.0mg pepstatin in 5mL methanol ---> 1.2mM pepstatin | |||
<u>SDS-PAGE Sample Buffer (No DTT)</u> | |||
# 3.75mL 0.5M Tris pH 6.8 ---> 6.1042 g Tris in 100mL water ---> 0.504M Tris | |||
# 15.0mL of 50% glycerol ---> 7.5mL glycerol + 7.5mL water | |||
# 0.3ml of 1% Bromphenol Blue ---> 0.009g Bromphenol Blue in 1mL water | |||
# 6.0mL 10% Sodium Dodecyl Sulfate (SDS) ---> 1g SDS in 10mL water | |||
# H<sub>2</sub>O to make 30mL total | |||
==Notes== | ==Notes== |
Revision as of 11:44, 24 September 2013
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ObjectiveTo 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. DescriptionThe 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.
DataMaking the solutions Glycine-HCl Buffer pH 3
50 mL of 5 mg/mL Hemoglobin
Pepstatin 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)
NotesThis area is for any observations or conclusions that you would like to note.
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