Purification of IgG specific to full-length VAR2CSA ectodomains

Antigen coupling

• Prepare 1mg of the recombinant full-length VAR2CSA protein by overnight dialysis at 4°C in the coupling buffer
• Wash 1ml of NHS-activated Sepharose 4 Fast Flow resin with 10 medium volumes of cold 1 mM HCI immediate before use
• Mix the washed resin and the antigen (dialyzed in the coupling solution).
• Perform the coupling at 4°C overnight
• After the coupling is completed, block any non-reacted groups on the resin with 2ml of 0.5 M Ethanolamine, 0.5 M NaCl, pH 8.3 for 2 hours at room temperature.
• Wash the antigen-coupled resin after coupling by alternating Wash Buffer A and Wash Buffer B (high and low pH respectively). Use 3 x 1 resin volumes Wash Buffer A followed by 3 x 1 resin volumes Wash Buffer B. 
• Repeat the cycle 6 times.
• The VAR2CSA-coupled affinity resin ready is now ready for use. 
• Store at 4°C in 1ml storage solution

Affinity purification of IgG specific to VAR2CSA

• Wash 2X the VAR2CSA-coupled resin with 2ml of PBS (spin at 300g for 2 minutes)
• Add the Total IgG diluted in PBS to the washed antigen-coupled resin
• Incubate 2 hours at room temperature by shaking on a 3D orbital shaker
• Wash 3X the IgG-bound resin with PBS (spin at 300g for 2 minutes)
• Elute the bound IgG with IgG Elution buffer, pH 2.8 (Invitrogen, Carlsbad, CA)
• Neutralize the eluted IgG with 2M Tris buffer pH 9.0
• Dialyze the eluted fractions into PBS pH 7.4 overnight at 4°C
• Concentrate the eluted IgG using a 50KDa Spin column (Amicon)
• Quantify the purified IgG using a Nanodrop (ND2000, Thermo Fisher Scientific, Waltham, MA)

Assay Solutions

Coupling buffer: A standard buffer is 0.2 M NaHCO3, 0.5 M NaCl, pH 8.3

Wash Buffer A (High pH): 0.1 M Tris-HCl, 0.5 M NaCl, pH 9

Wash Buffer B (Low pH): 0.1 M Acetate, 0.5 M NaCl, pH 4.5

Storage solution: 0.05%NaN₃ in PBS

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