Feb 23, 2022
Version 2
Cross-linking of IgG to Protein A or G Beads (S1425/S1430) V.2
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
External link: https://www.neb.com/protocols/0001/01/01/cross-linking-of-igg-to-protein-a-or-g-beads
Protocol Citation: New England Biolabs 2022. Cross-linking of IgG to Protein A or G Beads (S1425/S1430). protocols.io https://dx.doi.org/10.17504/protocols.io.bhkwj4xeVersion created by Julia Rossmanith
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 18, 2020
Last Modified: February 23, 2022
Protocol Integer ID: 38262
Keywords: Protein A, Protein G, cross-linkage, cross linked, covalent cross-linking of the IgG to protein A/G, igG purification to beads
Abstract
This protocol consists of an IgG purification step followed by covalent cross-linking of the IgG to the Protein A/G solid support. For IgG that has been previously purified, proceed directly to the cross-linking protocol.
Guidelines
Overview
This protocol consists of an IgG purification step followed by covalent cross-linking of the IgG to the Protein A/G solid support. For IgG that has been previously purified, proceed directly to the cross-linking protocol.
Materials
MATERIALS
Protein A Magnetic Beads - 1 mlNew England BiolabsCatalog #S1425S
Protein G Magnetic Beads - 1 mlNew England BiolabsCatalog #S1430S
Materials Needed:
- Elution Buffer: 0.1 M glycine-HCl (pH 2.5)
- Binding Buffer: 0.1 M NaPhosphate Buffer (pH 8.0)
- Dimethyl pimelidate dihydrochloride (Sigma, D-8388) dissolved at 25 mM in Cross-linking Buffer.
- Cross-linking Buffer: 0.2 M triethanolamine (pH 8.2)
- Blocking Buffer: 0.1 M ethanolamine (pH 8.2)
- 10x Phosphate Buffered Saline Gibco, ThermoFisherCatalog #70011044
- Tween 20Sigma AldrichCatalog #P9416-50ML
- Sodium azideSigma AldrichCatalog #71289
- Immunoglobulin in Binding Buffer
- 100 μl PBS, 0.1% Tween 20, 0.02% sodium azide
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
The IgG Purification protocol is for the binding of 20 µg purified IgG or isolation of 20 µg IgG from serum .
IgG Immobilization
IgG Immobilization
Note
The IgG immobilization protocol is for the binding of 20 µg purified IgG or isolation of 20 µg IgG from serum .
Vortex and thoroughly resuspend Protein A Magnetic Beads.
Aliquot 100 µL bead suspension to a sterile microcentrifuge tube.
Add 500 µL 0.1 M NaPhosphate Buffer (pH 8.0) and vortex to resuspend. Apply magnet for 00:00:30 , to pull beads to the side of the tube and remove supernatant. (Wash 1/2)
Repeat wash: Add 500 µL 0.1 M NaPhosphate Buffer (pH 8.0) and vortex to resuspend. Apply magnet for 00:00:30 , to pull beads to the side of the tube and remove supernatant. (Wash 2/2)
Add to the beads 80 µL 0.1 M NaPhosphate Buffer (pH 8.0) .
Add 15 µL -25 µL serum OR 20 µg purified IgG in a maximum volume of 30 µL .
Mix thoroughly and incubate at 4 °C with agitation for 00:30:00 .
Apply magnet and remove supernatant.
Add 500 µL 0.1 M NaPhosphate Buffer (pH 8.0) and vortex to resuspend. Apply magnet for 00:00:30 , to pull beads to the side of the tube and remove supernatant. (Wash 1/3)
Add 500 µL 0.1 M NaPhosphate Buffer (pH 8.0) and vortex to resuspend. Apply magnet for 00:00:30 , to pull beads to the side of the tube and remove supernatant. (Wash 2/3)
Add 500 µL 0.1 M NaPhosphate Buffer (pH 8.0) and vortex to resuspend. Apply magnet for 00:00:30 , to pull beads to the side of the tube and remove supernatant. (Wash 3/3)
IgG Cross-linking to Protein A/G Magnetic Beads
IgG Cross-linking to Protein A/G Magnetic Beads
30m
30m
Add 1 mL Cross-linking Buffer (0.2 M triethanolamine, [pH 8.2]) to the Protein A/G immobilized antibody.(Wash 1/2)
Note
At this point the purified IgG can be eluted from the beads or used directly for immunoprecipitation of target proteins. The purified IgG can also be cross-linked to the Protein A beads (see cross-linking protocol) to create a reusable immunoprecipitation bead which prevents the co-elution of antibody with target protein.
Vortex to resuspend. (Wash 1/2)
Apply magnet for 00:00:30 , to pull beads to the side of the tube and remove supernatant. (Wash 1/2)
Add 1 mL Cross-linking Buffer (0.2 M triethanolamine, [pH 8.2]) to the Protein A/G immobilized antibody.(Wash 2/2)
Vortex to resuspend. (Wash 2/2)
Apply magnet for 00:00:30 , to pull beads to the side of the tube and remove supernatant. (Wash 2/2)
Resuspend in 1 mL Cross-linking Buffer containing 25 millimolar (mM) DMP (6.5 mg DMP/ml of buffer) .
Mix thoroughly and incubate at Room temperature for 00:45:00 with agitation.
Apply magnet for 00:00:30 , to pull beads to the side of the tube and remove supernatant.
Add 1 mL Blocking Buffer (0.1 M ethanolamine, [pH 8.2]) .
Vortex to resuspend.
Apply magnet for 00:00:30 , to pull beads to the side of the tube and remove supernatant.
Add 1 mL Blocking Buffer .
Vortex to resuspend.
Incubate for 00:30:00 at Room temperature with agitation.
30m
Apply magnet for 00:00:30 , to pull beads to the side of the tube and remove supernatant.
Add 1 mL PBS . (Wash 1/3)
Vortex to resuspend. (Wash 1/3)
Apply magnet for 00:00:30 , to pull beads to the side of the tube and remove supernatant. (Wash 1/3)
Add 1 mL PBS . (Wash 2/3)
Vortex to resuspend. (Wash 2/3)
Apply magnet for 00:00:30 , to pull beads to the side of the tube and remove supernatant. (Wash 2/3)
Add 1 mL PBS . (Wash 3/3)
Vortex to resuspend. (Wash 3/3)
Apply magnet for 00:00:30 , to pull beads to the side of the tube and remove supernatant. (Wash 3/3)
Add 1 mL Elution Buffer (0.1 M glycine-HCl [pH 2.5]) .
Vortex to resuspend.
Apply magnet for 00:00:30 , to pull beads to the side of the tube and remove supernatant.
Note
This elutes bound antibody that is not cross-linked with DMP.
Add 1 mL PBS . (1/2)
Vortex to resuspend. (1/2)
Apply magnet for 00:00:30 , to pull beads to the side of the tube and remove supernatant. (1/2)
30s
Add 1 mL PBS . (2/2)
Vortex to resuspend. (2/2)
Apply magnet for 00:00:30 , to pull beads to the side of the tube and remove supernatant. (2/2)
30s
Resuspend and store beads in 100 µL PBS , 0.1% Tween 20, 0.02% sodium azide.