User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/23
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Horseradish peroxidase was made by weighting out 0.004g of horseradish peroxidase power. The weighted out amount was mixed with 1mL of sterile water. Molecular weight of horseradish peroxidase is 66kDa. Calculation is depicted as follows: 0.004g X 66,000g/mol ÷ 0.001L = 9.2X10-6M = 9.2μM
Carbonate buffer was made by mixing 100mL of 1M sodium bicarbonate with 100mL of 1M sodium bicarbonate together. pH of carbonate buffer was diluted by adding 1M and 6M HCl into the solution For calculations done to obtain the amount of grams weighted out the sodium carbonate and sodium bicarbonate, please consult Melissa's Notebook.
Molecular weight of sodium carbonate: 105.9784g/mol 0.0106g X 105.9784g/mol ÷ 0.1L = 1X10-3M = 1mM sodium carbonate
Molecular weight of sodium bicarbonate: 84.007g/mol 0.0084g X 84.007g/mol ÷ 0.1L = 1X10-3M = 1mM sodium bicarbonate
The 100mL of 1M sodium bicarbonate is mixed with 100mL of 1M sodium carbonate in a 1000mL graduated cylinder. pH was taken with a pH meter and the pH turned out to be 10.5. Initially 1M HCl was added drop wise into the carbonate buffer mix. About 50mL of 1M HCl was added to carbonate buffer mix to adjust the pH to 9.5. Then, 6M HCl was added to speed up pH adjustment process. About 10mL of 6N HCl was added drop wise to adjust the pH to 8.5. Please refer to Melissa's Notebook for calculations on determining the new molar concentration of carbonate buffer. The final concentration of carbonate buffer is calculated to be 0.833mM. Thus, 256mL of 0.833mM carbonate was made for luminol assay the next day.