Conjugation of a nanoCLAMP to SulfoLink™ resin

Abstract

Preparation of a nanoCLAMP affinity resin by coupling nanoCLAMP(Cys) with a unique C-terminal cysteine to SulfoLink™ agarose beads via a thioether linkage

Instructions

1	Determine the amount of nanoCLAMP(Cys) solution needed to make the desired volume of SulfoLink™ resin Use the eluate from "Ni-NTA purification of denatured His-tagged nanoCLAMP proteins from NEc-1 cell pellets"
2	Adjust the protein concentration of the nanoCLAMP eluate to 8 mg/ml in "Denaturing IMAC Buffer - 250 mM Imidazole" If the starting material is too dilute, concentrate with an Amicon Ultracel 3K (3000 NMWL)
3	To reduce each nanoCLAMP's cysteine residue, add "TCEP, 0.5 M" to the protein solution for a final concentration of 2 mM and mix gently
4	Incubate protected from light for at least for 1 hour at Room Temperature Do not incubate longer than overnight
5	Gently swirl SulfoLink™ resin to resuspend Minimize foaming
6	Transfer the appropriate volume of SulfoLink™ resin to a 50 ml chromatography column. Use a volume of packed SulfoLink™ resin equal to half the volume of the 8 mg/ml nanoCLAMP solution. E.g. for 10 ml of 8 mg/ml nanoCLAMP solution, use 5 ml of packed SulfoLink™ beads.
7	Drain the resin to the bed surface Throughout the procedure, never allow the column to run dry
8	Add 1 bed volume of "Denaturing IMAC Buffer - EDTA," swirl or rock gently to wash, and drain the resin to the bed surface. Repeat this step for a total of 4 times Avoid bubbles and never allow the column to run dry
9	Add the solution of reduced nanoCLAMP and swirl or rock column gently
10	Rotate the mixture of reduced nanoCLAMP and resin for 30 minutes at Room Temperature
11	Stand the column upright for 15 minutes at Room Temperature
12	Drain the column to the bed surface Reserve the flow through in case subsequent analysis is needed
13	Add 1 bed volume of "Denaturing IMAC Buffer - EDTA," swirl or rock gently to wash, and drain the resin to the bed surface. Repeat this step for a total of 3 times If using a pump, minimize the head volume and use a flow rate of 1 to 2 ml/min.
14	To block unreacted groups, add 2 bed volumes of freshly prepared "L-Cysteine pH 8.5, 50 mM," swirl or rock gently to wash
15	Rotate the mixture of reduced nanoCLAMP and resin for 30 minutes at Room Temperature
16	Stand the column upright for 15 minutes at Room Temperature
17	Drain the resin to the bed surface
18	Add 1 bed volume of "Denaturing IMAC Buffer," swirl or rock gently to wash, and drain the resin to the bed surface. Repeat this step for a total of 4 times
19	Add 1 bed volume of "MBS pH 6.5," swirl or rock gently to wash, and drain the resin to the bed surface. Rinse the walls of the column well.
20	Add 1 bed volume of "MBS pH 6.5 - CaCl₂," swirl or rock gently to wash, and drain the resin to the bed surface. . Repeat this step for a total of 4 times
21	Cap the bottom of the column and add 1 bed volume of "MBS pH 6.5 - CaCl₂" to make a slurry
22	Cut the tip off of a sterile transfer pipet with a fresh razor blade
23	Use the pipet to transfer the slurry to a 15 ml or 50 ml conical tube on ice. Rinse the column with a small amount of buffer to maximize yield.
24	Centrifuge at 1,000 X g for 5 minutes at 4°C
25	Estimate volume of resin by eye and adjust to a 50% (vol:vol) slurry
26	Store for up to 6 months at 4°C

Solution(s) Used

Solution	Usage Rate
Denaturing IMAC Buffer - 250 mM Imidazole
Denaturing IMAC Buffer - EDTA	7 ml per ml of packed resin
L-Cysteine pH 8.5, 50 mM	2 ml per ml of packed resin
Denaturing IMAC Buffer	4 ml per ml of packed resin
MBS pH 6.5	2 ml per ml of packed resin
MBS pH 6.5 - CaCl2	4 ml per ml of packed resin

Ingredient(s) Used

Ingredient	Usage Rate
TCEP HCl, 0.5 M	2 ul per ml of nanoCLAMP solution

Outside Material(s) Used

Vendor	Outside Material	Catalog Number	Usage Rate
ThermoFisher Scientific	SulfoLink™ Coupling Resin	20401	0.5 ml packed resin per ml of 8 mg/ml nanoCLAMP solution