Cross-linking of IgG to Protein A or G Beads (S1425/S1430)
Vortex and thoroughly resuspend Protein A Magnetic Beads. 
Aliquot 100 μl of bead suspension to a sterile microcentrifuge tube (wash #1). 
Add 500 μl 0.1 M NaPhosphate Buffer (pH 8.0). 
(wash #1) Vortex to resuspend. 
(wash #1) Apply magnet for 30 seconds, to pull beads to the side of the tube. 
(wash #1) Remove supernatant. 
(wash #2) Add 500 μl 0.1 M NaPhosphate Buffer (pH 8.0). 
(wash #2) Vortex to resuspend. 
(wash #2) Apply magnet for 30 seconds, to pull beads to the side of the tube. 
(wash #2) Remove supernatant. 
Add to the beads 80 μl of 0.1 M NaPhosphate Buffer (pH 8.0). 
Add 15-25 μl of serum OR 20 μg purified IgG in a maximum volume of 30 μl. 
Mix thoroughly and incubate at 4°C with agitation for 30 minutes. 
Apply magnet and remove supernatant.
(wash #1) Add 500 μl 0.1 M NaPhosphate Buffer (pH 8.0). 
(wash #1) Vortex to resuspend. 
(wash #1) Apply magnet for 30 seconds, to pull beads to the side of the tube. 
(wash #1) Remove supernatant. 
(wash #2) Add 500 μl 0.1 M NaPhosphate Buffer (pH 8.0). 
(wash #2) Vortex to resuspend. 
(wash #2) Apply magnet for 30 seconds, to pull beads to the side of the tube. 
(wash #2) Remove supernatant. 
(wash #3) Add 500 μl 0.1 M NaPhosphate Buffer (pH 8.0). 
(wash #3) Vortex to resuspend. 
(wash #3) Apply magnet for 30 seconds, to pull beads to the side of the tube (wash #3) Remove supernatant. 
Add 1 ml of Cross-linking Buffer (0.2 M triethanolamine, [pH 8.2]) to the Protein A/G immobilized antibody. 
(wash a) Vortex to resuspend. 
(wash a) Apply magnet for 30 seconds, to pull beads to the side of the tube. 
(wash a) Remove supernatant. 
(wash a) Add 1 ml of Cross-linking Buffer (0.2 M triethanolamine, [pH 8.2]) to the Protein A/G immobilized antibody. 
(wash b) Vortex to resuspend. 
(wash b) Apply magnet for 30 seconds, to pull beads to the side of the tube.  
(wash b) Remove supernatant. 
(wash b) Resuspend in 1 ml Cross-linking Buffer containing 25 mM DMP (6.5 mg DMP/ml of buffer). 
Mix thoroughly and incubate at room temperature for 45 minutes with agitation. 
Apply magnet for 30 seconds, to pull beads to the side of the tube. 
Remove supernatant. 
Add 1 ml Blocking Buffer (0.1 M ethanolamine, [pH 8.2]).   
Vortex to resuspend.  
Apply magnet for 30 seconds, to pull beads to the side of the tube. 
Remove supernatant. 
Add 1 ml of Blocking Buffer Vortex to resuspend. 
Incubate for 1 hour at room temperature with agitation. 
Apply magnet for 30 seconds, to pull beads to the side of the tube. 
Remove supernatant. 
(wash #1) Add 1 ml of PBS. 
(wash #1) Vortex to resuspend. 
(wash #1) Apply magnet for 30 seconds, to pull beads to the side of the tube. 
(wash #1) Remove supernatant. 
(wash #2) Add 1 ml of PBS. 
(wash #2)  Vortex to resuspend. 
(wash #2) Apply magnet for 30 seconds, to pull beads to the side of the tube (wash #2) Remove supernatant. 
(wash #3) Add 1 ml of PBS. 
(wash #3)  Vortex to resuspend. 
(wash #3)  Apply magnet for 30 seconds, to pull beads to the side of the tube. 
(wash #3) Remove supernatant. 
Add 1 ml Elution Buffer (0.1 M glycine-HCl [pH 2.5]). 
Vortex to resuspend. 
Apply magnet for 30 seconds, to pull beads to the side of the tube. 
Remove supernatant. 
Resuspend and store beads in 100 μl PBS, 0.1% Tween 20, 0.02% sodium azide
