Affinity purification from E.coli lysates using nanoCLAMP(Resin)


This protocol describes the small scale batch, one step affinity purification of target proteins from E.coli whole cell lysates using nanoCLAMP(Resin). Target proteins are eluted under native conditions with neutral polyol elution buffer. Yields depend on the resin and targets involved, but typically range from 100 to 500 ug of >95% pure protein when target protein is not limiting.


1 Transfer 100 ul of 50% slurry of nanoCLAMP(Resin) to a small chromatography column
2 Wash resin with 500 ul PBS. Repeat this step for a total of 3 times
3 Cap bottom of column; add cleared lysate containing the nanoCLAMP antigen; and cap top of column
4 Incubate, rotating for 1 hour at 4°C
5 Drain column without letting resin run dry
Subsequent steps may be performed at room temperature
6 Wash resin with 500 ul PBS or PBS-T with up to 0.05% Tween 20. Repeat this step for a total of 4 times
7 Elute bound protein with 100 ul PEB. Repeat this step for a total of 4 times
More elutions may be required to remove all protein
8 To regenerate column, wash with 500 ul DB. Repeat this step for a total of 4 times
9 Wash with 500 ul MBS
10 Wash with MBS + 1 mM CaCl2. Repeat this step for a total of 4 times
CaCl2 may not be necessary but has not been tested
11 Add azide to 0.05% and store at 4°C

Solution(s) Used
Use / Nickname Solution
MBS MBS pH 6.5
Polyol Elution Buffer PEB
Applications Using This Protocol