Transferration of the cell suspension into the celldisrupter and squash three times at 2.7 kbar
Centrifugation at 13.000 rpm at 4°C for an hour
continue with the protocol IF YOU HAVE an histag in your protein
Load the prepared IMAC column with the hole supernatant and collect the flowthrough
Wash the column two times with 20 mM imidazole in PBS buffer pH 7.4 and collect the floqthrough
Wash the column in five steps with increasing imidazole concentrations (40mM, 60mM, 100mM, 200mM and 500mM) to elute the protein and collect the flowthrough
Load the collected flowthrough on a 12% SDS-PAGE (Schägger)
Combine the collected passages of the right protein and concentrate it to an amont of 5 mL
To get rid of waste and undesired proteins perform a gelfiltration with FPLC (fast protein liquid chromatography)
Usage of HiLoad 16/60 Superdex 75g from GE Healthcare
Equilibration of the column with PBS buffer pH 7.4
Loading 5 mL of the protein solution
Elute the protein with pure PBS buffer pH 7.4 and collect the samples
measure the protein concentration
aliquot in 1 mL stocks and freeze in liquid nitrogen
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