User:Matt Hartings/Notebook/Photosynthesis/2013/03/01

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FPLC

Running FPLC in order to purify the Mn-Hb and the Ni-Hb.

Here is a picture of the two variants prior to purification. (The Mn-Hb is in the more orange liquid and the Ni-Hb is in the more maroon liquid)

For the purification, I am using the following buffers:

Binding: 10mM Tris, 50mM NaCl, pH 8.5 Elution: 10mM Tris, 1M NaCl, pH 8.5

The Mn-Hb proceeded in a similar fashion to the WT-Hb, eluting at around 10% BufferB.

The Ni-Hb was not purified with these conditions. Something stuck to the column, which there was much that passed through the column. The portion that stuck to the column could not be eluted with the elution buffer. I ran all of the solution through the column and collected the flowthru.


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