SulfoLink affinity purification of antibodies

·SulfoLink affinity purification

Introduction

Affinity purification protocol for antibodies from sera. The protocol can be used for antibodies generated against Cys-containing peptides. It requires the serum, ~2 mg of the Cys-containing peptide used for immunisation and some custom solutions and plastic components.

Materials 

Reagents

-serum of final bleed after immunisation

-Cys-containing peptide

-SulfoLink Coupling Resin (Thermo Fisher, 20401)

Solutions

-Coupling Buffer: 50 mM Tris pH 8.5, 5 mM EDTA (50 ml per antibody)

-12.1 mg Cys, dissolved fresh in 2 ml coupling buffer before use

-1 M NaCl (30 ml per antibody)

-PBS (300 ml per antibody)

-0.5 M NaCl in PBS (15 ml per antibody)

-100 mM glycine pH 2.7 (10 ml per antibody)

-100 mM glycine pH 2.3 (10 ml per antibody)

-1 M Tris, pH 9.5

-5M MgCl2 (10 ml per antibody)

Components

-chromatography column (e.g. Bio-Rad Econo-Pac® Chromatography Columns for gravity-flow chromatography)

-0.2 μM filter with syringe

-Vivaspin® Turbo 4, 10 kDa, PES, 25 pc or similar

-rotating mixer

Protocol

Column preparation

-Install the chromatography column on a stand at room temperature

-Clean column several times with distilled water, rinse it with 70% ethanol

-Mix 2 ml of Sulfolink coupling gel (Thermo Scientific, Rockford, USA) with 10 ml of Coupling Buffer (50 mM Tris pH 8.5, 5 mM EDTA) in the closed chromatography column, invert a few times to equilibrate gel (2 ml of coupling gel beads correspond to about 5 ml of beads in solution from the stock bottle)

-Open the bottom of the column to remove Coupling Buffer

-Dissolve fresh 2 mg of Cys-containing peptide in 2 ml of Coupling Buffer

-Add 2 ml peptide solution and rotate the column at room temperature for 15 min

-Incubate at room temperature for 30 min without mixing

-Open the column and remove buffer

-Optional: (if peptide contains aromatic amino acid): check peptide concentration (A280) and compare to original peptide solution to determine coupling efficiency

-Wash the beads 3 times with 10 ml Coupling Buffer

-Add 2 ml of 50 mM cysteine in Coupling Buffer (made fresh, 12.1 mg Cys into 2 ml) and rotate for 15 min at room temperature to quench unreacted sites

-Incubate at room temperature for 30 min without mixing

-Wash 3 times with 10 ml 1 M NaCl (about 5 min each)

-Wash 3 times with 25 ml PBS

Antibody binding

-Defrost the antibody serum, and filter through a 0.2 μM filter (this step will increase flow through speed)

-Add the serum to the peptide-coupled beads in the column, close with the lid. For rabbit sera, use half of the volume and freeze the other half, for sera from smaller animals you can use all of the serum. If the final volume is larger than the volume of your column, transfer to a 50 ml Falcon tube.

-Mix overnight in the cold room on a rotating mixer

-Put the column back to the holder and wait 15 min until the resin sediments (at room temperature). If you used a Falcon tube, add the mixture back to the column in steps, let the resin sediment and the serum to flow through

-Open the column and allow all the serum to flow through into a Falcon Tube (at room temperature)

-Run the flow-through once more through the column

-Wash the column 5 times with 25 ml PBS (these washing steps will take time, the flow rates can be low)

-Wash the column with 15 ml 0.5 M NaCl in PBS to remove weakly bound antibodies

-Wash the column twice with 10 ml PBS

Manual elution

-Prepare the following solutions, adjust the pH precisely with a pH meter (not pH strips):

-100 mM glycine pH 2.7

            100 mM glycine pH 2.3

-Elute 8x1 ml fractions with 100 mM glycine pH 2.7, into Eppendorf tubes containing 40 μl 1 M Tris, pH 9.5

-Elute 8x1 ml fractions with 100 mM glycine pH 2.3, into Eppendorf tubes containing 75 μl 1 M Tris, pH 9.5

-Elute 8x1 ml fractions with 5M MgCl₂, into empty Eppendorf tubes (this step will denature the antibodies and elute even the highest affinity ones)

-Measure protein concentration in each fraction using a Nanodrop machine (Thermo Scientific) or other method

-Pool fractions eluted with the same buffer. For the pH 2.7-eluted fractions pool only the fractions starting with the peak fraction, i.e. discard the left most side of the peak, but include the right side minor fractions, which presumably have a higher affinity. For the pH 2.3 and MgCl₂ eluted fractions pool everything with protein (it is expected that the progressively lower pH will elute progressively higher affinity antibodies, but in lower quantities).

-Concentrate the fractions for the glycine pH 2.7, pH 2.3, and MgCl₂ pooled fractions using Sartorius Vivaspin spin columns with 10,000 cut off, spin at 4,000 g for 5-40 min at 4 °C (the optimal time to spin will depend on your concentration - you need to check every 5-10 min and be careful not to dry it - leave at least 100-200 μl solution)

-Add to the concentrated fraction 10-20 fold 50 % glycerol-PBS solution and spin down again to a final volume of 100-200 μl

-Measure protein concentration in the concentrated samples using the Nanodrop or other method

Antibody storage

-Dilute antibodies to 50% glycerol (final concentration) and store aliquots at -20°C for mid-term storage (1-2 years) or -70°C for long-term storage

Notes

One critical step in the protocol is getting the Tris and glycine solutions right during elution. We elute the antibodies with low pH glycine and immediately neutralise them by collecting into tubes containing 1M Tris at pH 9.5. Well before the elution step, prepare and the glycine and Tris solutions as accurately as possible and run tests of how much Tris you need to neutralize the glycine - it should be about 40 μl for 950 μl of glycine ph 2.7, but this may vary depending on your solutions You can use pH indicator strips to test the ratio to obtain a final pH of 7.5-8.