User:Madeleine Y. Bee/Notebook/CHEM-571 2013F/2013/09/24

Experimental Biological Chemistry: Fall 2013

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September 24, 2013 Objective Pepsin cleavage of hemoglobin according to this procedure. Description Combine 5 mL of the hemoglobin solution with the amount of pepsin to make the final concentration 2 nM Do the same only with an amount of pepstatin to make the final concentration 20nM Incubate these solutions at 37C After 30 minutes, remove two samples: SDS-PAGE Samples (for TOMORROW) 10uL of the reaction sample, diluted to 1mL with the Glycine-HCl buffer 10uL of this diluted sample, mixed with 10uL of the SDS-PAGE running buffer Store all of your SDS-PAGE samples in the fridge UV-Vis Samples (for TODAY) 0.75mL of the reaction sample, add 0.75mL of 1M perchloric acid to precipitate the remaining hemoglobin After 1 hour, centrifuge for 15 minutes to remove solid (uncleaved hemoglobin) from solution Measure the absorption (specifically note 280nm) to determine the protein concentration in solution Use Glycine-HCl buffer as blank Use Hemoglobin as reference Repeat Step 3 every 30 minutes for 2 hours. Data Notes Gel electrophoresis will be conducted tomorrow.