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Purifying Hemoglobin - Andrew Castleman Purifying...

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Andrew Castleman 2/7/11 Purifying Hemoglobin By using a negative column then a positive column, the hemoglobin can be separated from other solutes left after using lysate in a sample of tadpole blood. To define the purity of the hemoglobin it should be based on the ratio of hemoglobin concentration to total protein concentration. By preparing a portion of each fraction by adding HCl protein dye, which is blue in water but brown in HCl and redevelops blue color in proportion of the total protein concentration, the absorbance at 620nm will indicate the total protein concentration. Another portion of each fraction can be diluted to the same volume and tested for absorbance at 410nm. In order to slowly elute the hemoglobin, a gradient maker made of two columns with a valve between them, can be used. When the valve is open the two columns will both feed towards the column so that the eluent is diluted as it enters the outflow column.
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