User:Matt Hartings/Notebook/Photosynthesis/2013/09/30

Protein Re-engineering

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Objective Take kinetic data for Mb catalyzed o-phenylenediamine + H2O2 reaction at low temperatures (5, 15, and 25C) Note - I'm only going to be able to take 5 and 15 today due to my first 15C data only saving two points (I was away at a meeting during this time) Procedure and Data Stock solutions As noted on Friday, OPD will oxidize in water over time. OPD and H2O2 solutions will be made fresh for each run! sample 1 Stock solutions: Myoglobin 1.4mg (Mb:KCl lyophilized from Citrate, 5mg:500mg) in 1.0mL water OPD 2.4mg OPD in 2.4mL water H2O2 0.114mL 30% H2O2 in 0.887mL water Experimental Run Sample chamber set to 5C and to stir N2 run through the cuvette holder to reduce condensation on cuvette 2.025mL OPD stock solution 0.75mL H2O2 stock solution Start Data Collection 1ms Integration 10 scan average Wait 1 minute after each scan Stop after 2 hours Files saved as: Mb_OPD_H2O2_Citrate_H20_5_SCANNUMBER.txt 0.225mL Mb stock solution added just before the third scan This is denoted in the data file for the third scan sample 2 Stock solutions: Myoglobin used same solution as above (1.4mg (Mb:KCl lyophilized from Citrate, 5mg:500mg) in 1.0mL water) OPD 3.3mg OPD in 3.3mL water H2O2 0.114mL 30% H2O2 in 0.887mL water Experimental Run Sample chamber set to 15C and to stir N2 run through the cuvette holder to reduce condensation on cuvette 2.025mL OPD stock solution 0.75mL H2O2 stock solution Start Data Collection 1ms Integration 10 scan average Wait 1 minute after each scan Stop after 2 hours Files saved as: Mb_OPD_H2O2_Citrate_H20_15_SCANNUMBER.txt 0.225mL Mb stock solution added just before the third scan This is denoted in the data file for the third scan Note only two data points saved. I think I entered stop after 2 minutes instead of stop after 2 hours. Need to run again sample 3 Stock solutions: Myoglobin used same solution as above (1.4mg (Mb:KCl lyophilized from Citrate, 5mg:500mg) in 1.0mL water) OPD 3.7mg OPD in 3.7mL water H2O2 0.114mL 30% H2O2 in 0.887mL water Experimental Run Sample chamber set to 15C and to stir N2 run through the cuvette holder to reduce condensation on cuvette 2.025mL OPD stock solution 0.75mL H2O2 stock solution Start Data Collection 1ms Integration 10 scan average Wait 1 minute after each scan Stop after 2 hours Files saved as: Mb_OPD_H2O2_Citrate_H20_15_SCANNUMBER.txt 0.225mL Mb stock solution added just before the third scan This is denoted in the data file for the third scan