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

Abstract

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.

Instructions

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
PBS-T	PBST RJS
PBS	PBS RJS
MBS	MBS pH 6.5
DB	DB
Polyol Elution Buffer	PEB

Applications Using This Protocol

Outcome	Title
Successful	SUMOstar affinity purification with nanoCLAMP SMT3-A1(Resin), batch mode from E. coli lysate
Successful	Thioredoxin purification by affinity chromatography with nanoCLAMP trxA-A1(Resin), batch mode from E. coli lysate
Successful	mCherry purification by affinity chromatography with nanoCLAMP mCher-A2(Resin), batch mode from E. coli lysate
Successful	Beta-galactosidase purification by affinity chromatography with nanoCLAMP lacZ-A2(Resin), batch mode from E. coli lysate
Successful	Avidin purification by affinity chromatography with nanoCLAMP AVD-A1(Resin), batch mode with E. coli lysate
Successful	SUMO purification by affinity chromatography with nanoCLAMP SMT3-A1(Resin), batch mode from E. coli lysate
Successful	NusA purification by affinity chromatography with nanoCLAMP nusA-A1(Resin), batch mode from E. coli lysate
Successful	MBP purification by affinity chromatography with nanoCLAMP malE-A1(Resin), batch mode from E. coli lysate
Successful	GFP purification by affinity chromatography with nanoCLAMP GFP-A1(Resin), batch mode from E. coli lysate
Successful	Purification of neutravidin from E. coli lysate by batch affinity chromatography with AVD-A1(Resin)