Protocols

 

 

Conjugation of GX41 antibody to resin (Duration: 8 hours ~ 1 day)

 

Material needed:

  • Affi-gel 10 resin (Bio-Rad, cat. no. 153-6046)
  • Anti-diglycyl-lysine, GX41 antibody (Lucerna, cat. no. 30-1000 or 30-0100
  • Hydrochloric acid (Fisher, cat. no. A144-500)
  • Phosphate buffered saline (Fisher, cat. no. BP399-1)
  • Sodium azide (ICN Biomedicals, cat. no. 102891)
  • Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, Invitrogen, cat. no. 15504-20)
  •  4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Invitrogen, cat. no. 11344-041)

 

Procedure:

1.     Transfer 50 µL of 50 % Affi-gel 10 resin to a 1.6 mL Eppendorf tube.

2.     Wash Affi-gel 10 resin twice with 1 mL of ice-cold 1 mM HCl.

3.     Adjust pH of 200 µL of GX41 antibody (~1 mg/mL) to ~8.5 with 1 N HCl and buffer with 50 µL 1 M HEPES [8.5] (final concentration = 0.2 M).

4.     Add antibody solution to Affi-gel 10 resin and incubate at 25 °C for 4 h or at­ 4 °C overnight.

5.     Wash beads with 500 µL of 1 M Tris-HCl (pH 8.0) and incubate with 500 µL of 1 M Tris-HCl solution on an end-over-end rotator at 25 °C for 2 h or at 4 °C overnight.

6.     Briefly wash beads twice with 500 µL of 0.1 M glycine (pH 2.7), and twice with 0.1 M Tris-HCl (pH 8.0) to neutralize pH.

7.  Resuspend Affi-gel 10 resin with PBS containing 0.02 % sodium azide and store at 4°C.


Notes:

1. Larger batches can be obtained by increasing the quantity of all components proportionally.  

2. Alternatively, protocols of coupling antibody to protein G beads can be used here.

3. The immobilized antibody can be kept at 4 °C for more than four weeks.

 

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