Contaminant depletion using nanoCLAMP(Resin)

Abstract

This protocol is for depleting contaminating proteins from purified protein preparations using nanoCLAMP(Resin) specific for the contaminant. The resin is incubated in excess of the contaminant protein and the target protein is collected in the flow through. The resin can be purged of the contaminant by regenerating with GuHCl and re-used several times. Estimate the amount of contaminant in the protein sample prior to depletion by densitometry and use the nanoCLAMP(Resin) working binding capacity, which can be found on the product page on the website, to determine the amount of resin to add to bind at least 2X the amount of contaminant. This protocol can be scaled as necessary.

Instructions

1	Transfer 20 ul nanoCLAMP(Resin) (50% slurry) to a small chromatography column
2	Wash resin with 100 ul PBS and drain buffer to just above resin bed. Repeat this step for a total of 3 times
3	Cap the bottom of the column and add 50 ul of protein contaminated with target of nanoCLAMP(Resin)
4	Cap the column and rotate for 1 hour at 4°C
5	Allow resin to settle, uncap the column, and collect the target protein in the flow-through
6	To regenerate resin, wash with 100 ul DB. Repeat this step for a total of 4 times
7	Wash with 100 ul MBS
8	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
9	Add azide to 0.05% and store at 4°C

Solution(s) Used

Use / Nickname	Solution
PBS	PBS RJS
MBS	MBS pH 6.5
MBS + CaCl2	MBS pH 6.5 - CaCl2
DB	DB

Applications Using This Protocol

Outcome	Title
Successful	Direct removal of SlyD contaminant from IMAC eluates using SlyD-A1(Resin)
Successful	SlyD removal from Ni-NTA purified protein with nanoCLAMP slyD-A1(Resin)