Friday, June 23, 2006

Purification and SDS-PAGE

I harvested after about 18 hours. Both cultures were clean and saturated. I used 5 mls of a culture in Sigma's His-Select iLap 5 ml purification column. The column is disposable and has only three steps: lyse, wash and elute.

I didn't order the required wash and elute buffers from Sigma because they were $150 and 100x more than I needed; so I made the buffers. Bad decision partly: my pH meter wasn't calibrating correctly, so I had to use a buffer calculator to figure the recipe with a fixed ionic strength. Something must have happend there, because the protein came out in the wash instead of the elute. At that, the corrosponding band in the cellular lysate was more intense than the "purified" band. But it was there, at the correct weight of 47kDa (actual is 46,562 Da).

Details on the SDS-PAGE: I ran the elution, a second elution (not suggested by Sigma), the wash flow-through, the lysed cell flow-through and the lysed cells (not the flow-trhough) on a 4-15% gradient Ready Gel from Bio-Rad courtesy of Ms. Marietta Dunaway. I used Novagen's Perfect Protein ladder 15-150 kDa. (The ladder is nice because it contains no oligosaccharides.)


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