Antibody purification

Exchange the buffer of the MBP fusion protein using pD10 columns

• Equilibration buffer:

100 mM HEPES pH 8.0 (shelf!) 500 mM NaCl (2.922g NaCl for 100 ml; M=58.44g/mol) 10 mM Maltose (M=360.32g/mol; shelf: 20g/100ml⇒ 20%) Add CaCl2 to 80 mM final conc. Save an aliquot for later analysis ⇒ Add in a later stage!

• Buffer exchange:

equilibrate PD10 column with 4 CV (column volumes) 2.5 ml of protein solution (if less, fill it up to 2.5ml with buffer) Elute protein with 3.5 ml of equilibration buffer Add CaCl2 to 80 mM final conc. to protein solution

• Cleaning of PD10 columns:

Wash with minimum 4 CV 1x PBS Wash with minimum 3 CV 10x PBS Store the columns in 10x PBS at 4 C

Affinity purification of antiserum on antigen coupled NHS HiTrap columns (Drechsel, A. Desai)

Solutions (sterile filtered):

- 1mM HCL (prepare fresh; shelf:1M)

- 0.1 M M glycine pH 2.6

- 0.1 M triethylamine pH 11.5 (δ=0.728g/l)

 (13.89 ml for 1l of solution)

- 2 M Tris pH 7.0 (or 100µl Tris pH 9.0 + 900µl Glycin, check pH! ) - 10 mM Tris pH 8.0

- 10x PBS

- 2x PBS

- 20% NaN3

- Blocking buffer 0.5 M ethanolamine (3.06 ml 100% EtOHamine for 100ml solution) 0.5 M NaCl

- Wash buffer 1x PBS 0.5 M NaCl 0.1% Triton X 100 (=10 ml Triton X-100 pure)

- Storage buffer 1x PBS (from 10x stock) 55% glycerol (v:v)

Flow rate: 1.5 ml (orange/violet tubing, Gilson settings at 3.5) ⇒ For calculation!

Protocol for coupling of MBP fusion protein to NHS column:

• Use 1 ml NHS HiTrap columns (all volumes mentioned are according to this size column!)

1. Wash with 1 mM HCl (ice cold, prepare fresh) for 2 min
2. Set-up antigen recirculation (5-8 ml of antigen (MBP fusion protein), 0.5 mg/ml protein, 2-10 mg total protein), recirculate for 30 min at RT
3. Block unreacted NHS groups with blocking buffer, flow for 4 min, stop flow and incubate for 30 min at RT
4. Wash with 10 mM Tris pH 8 for 5 min
5. Wash with 100 mM glycine pH 2.6 for 5 min
6. Wash with 10 mM Tris pH 8 for 5 min
7. Wash with 100 mM triethylamine pH 11.5 for 5 min
8. Wash with 10 mM Tris pH 8 for 5 min
9. Wash with 100 mM glycine pH 2.6 for 5 min
10. Wash with 10 mM Tris pH 8 for 5 min
11. Wash with 100 mM triethylamine pH 11.5 for 5 min
12. Wash with 1x PBS for 15 min

• Serum recirculation

1. Thaw serum
2. Add an equal volume of sterile 2x PBS and NaN3 to a final conc. of 0.1% (sterile filtered)
3. Recirculate the serum o/n at RT

First day!

• Wash and elution

1. Remove column from recirculation
2. Wash with wash buffer for 1 h (save a sample)
3. Wash with 1x PBS (save a sample
4. Add 100µl of 2 M Tris HCl pH 7.0 to 1.5 ml tubes in which the elution fractions are collected (40 tubes)
5. Wash with 12 ml of 10 mM Tris pH 8 (8 min)
6. Elute antibody with 20 ml of 0.1 M glycine pH 2.6 (13.5 min), collect 900µl in the prepared tubes, mix immediately to neutralize the antibody solution
7. Wash with 12 ml of 10 mM Tris pH 8 (8 min)
8. Elute antibody with 20 ml of 0.1 M triethylamine pH 11.5 (13.5 min), collect 900µl in the prepared tubes, mix immediately to neutralize the antibody solution
9. Wash with 12 ml of 10x PBS (8 min)
10. Wash with 12 ml of Storage buffer (8 min)
11. Label the column with the coupled protein name and store it at 4C°

• Determination of IgG concentration

- either by Bradford or using IgG program on NanoDrop - measure all fractions and pool the fractions with the antibody

Second day!

Dialyzing of antibody solution: - For dialyzing use e.g. Slide-A-Lyzer Dialysis Cassettes 10K (10000 MWCO) - hydrate the membrane in 1x PBS - use a grey neddle and remove the air from the cassette - Apply the antibody solution carefully into the cassette without damaging the membrane - Remove the air from the cassette -Apply a styropore clip to the cassette - Dialyze the antibody solution against minimum 3 changes of 1x PBS and finally against storage buffer at 4 C°

Aliquoting of antibody solution: - Prepare 50µl aliquots and store at 4C, -20C, -80C and lyophilized at -20C (has to be resuspend in 50µl water before use)