How to make anti-IgA beads for serum IgA purification

Preparation of anti-IgA beads

NHS-activated Sepharose 4 Fast Flow (NHS-beads): GE Healthcare

Goat polyclonal antibody Anti-mouse IgA: SouthernBiotech #1040-01, 1 mg/mL

12 mL of ice cold 1 mM HCl

↓+ 1 mL of NHS-beads slurry

↓cfg 1200xg 3 min

Pellet

↓+ 500 µL of 1 mg/mL goat polyclonal antibody anti-mouse IgA

pH check!! (Adjust the pH 6 to 9)

Incubate for 2 hr at RT (with gently shacking every 20 min)

↓+ 10 mL of 0.1 M Tris-HCl pH 8.0

↓cfg 1200xg 5 min

Pellet

↓+ 12 mL of 0.1 M Tris-HCl pH 8.0

Rotate in cold room for 6 hr to O/N

↓cfg 1200xg 5 min

(*1) 

Pellet 

↓+ 6 mL of 0.1 M acetate buffer, 0.5 M NaCl pH 4

↓cfg 1200xg 3 min

Pellet                                                     

↓+ 6 mL of 0.1 M Tris-HCl pH 8.0

↓cfg 1200xg 3 min

[Repeat (*1) 3 times]

↓

(*2) 

Pellet 

↓+ 12 mL of PBS with 0.05% Azide

↓cfg 1200xg 3 min

[Repeat (*2) 2 times]

↓

Pellet

↓+ 1 mL of PBS with 0.05% Azide ( = slurry volume is about 2 mL, anti-IgA conc. = 0.25 mg/mL)

Store at 4ºC = “anti-IgA beads”