Precipitator
Precipitator
Ruediger
inoculated cells from yesterdays plating. All 12 plates had colonies. I picked 2 from each plate.
Incubation at 37°C overnight at 300rpm.
further Project plan
- if GFP Pdb works, I will pick best affinity Pbd and use it for the precipitator.
- I will also need a primer for the Precipitator with a GST tag and a TEV cleavage site[http://en.wikipedia.org/wiki/TEV_protease] inbetween.
- Need to order a GST column (GST-4B) for the ÄKTA as well as PreScission Protease GST
- check toolbox plasmid for pGEX-6P
- check affinity calculation programs Scatchand plots and Klötz (to estimate Ni-Precipitator binding)
- data for these calculations could be generated with ultrafiltration of the protein bound to nickel, to see how much nickel goes through - can be tested chemically
- 300ml E.coli culture needed for protein purification in ÄKTA
- first there will be the GST affinity purification of the lysate of the E. coli culture -> Precipitator will be in the eluate
- second a digestion of the eluate with the Cleavage enzyme
- third another GST affinity purification -> Precipitator will be in the flowthrough
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